Large-scale epigenomic reprogramming links anabolic glucose metabolism to distant metastasis during the evolution of pancreatic cancer progression

ABSTRACT

The present invention relates to a method of identifying epigenetic reprogramming. Identifying epigenetic reprogramming comprises detecting large organized heterochromatin lysine (K)-9 modified domains (LOCKs) and large DNA hypomethylated blocks in a sample containing DNA from a subject having cancer, for example, PDAC.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 16/340,069 filed Apr. 5, 2019, now pending; which is a 35 USC § 371National Stage application of International Application No.PCT/US2017/055376 filed Oct. 5, 2017, now expired; which claims thebenefit under 35 USC § 119(e) to U.S. Application Ser. No. 62/405,155filed Oct. 6, 2016, now expired. The disclosure of each of the priorapplications is considered part of and is incorporated by reference inthe disclosure of this application.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under CA054358,CA140599, CA179991, CA180682 and CA095103 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

INCORPORATION OF SEQUENCE LISTING

The material in the accompanying sequence listing is hereby incorporatedby reference into this application. The accompanying sequence listing.xml file, named JHU4080-2_ST26.xml, was created on Jul. 19, 2023 and is55,580 bytes in size.

BACKGROUND OF THE INVENTION Field of the Invention

The invention relates generally to genetic analysis and morespecifically to cancer and the epigenetic influence on progression andmetastases of cancer.

Background Information

During the evolutionary progression of pancreatic ductal adenocarcinoma(PDAC), heterogeneous subclonal populations emerge that drive primarytumor growth, regional spread, distant metastasis, and patient death.However, the genetics of metastases largely reflects that of the primarytumor in untreated patients, and PDAC driver mutations are shared by allsubclones. This raises the possibility that an epigenetic process mightbe operative during metastasis. Here we detected striking epigeneticreprogramming of global chromatin modifications during the naturalevolutionary history of distant metastasis. Genome-wide mapping revealedthat these global changes were targeted to thousands of large chromatindomains across the genome that collectively specified malignant traits,including euchromatin and large organized chromatin K9-modified (LOCK)heterochromatin. Parallel to these changes, distant metastasesco-evolved a dependence on the oxidative branch of the pentose phosphatepathway (oxPPP), and oxPPP inhibition selectively reversed reprogrammedchromatin and blocked tumorigenic potential. Thus, divergent metabolic,epigenetic, and tumorigenic programs emerged during the evolution ofpancreatic cancer progression.

Despite significant progress in survival rates for most human cancers,PDAC remains nearly universally lethal with survival rates of 8%. Infact, PDAC is projected to be the second-leading cause of cancer deathsin the western world by 2020. Primary PDACs have been shown to containdistinct subclonal populations. However, these subclones share identicaldriver mutations and the genetics of metastases largely reflects that ofthe primary tumor. Furthermore, subclones are defined genetically bytheir unique progressor mutations, the vast majority if not all of whichare thought to be passenger events. This raises questions as to whatmechanisms might drive progression and metastasis during the naturalhistory of disease evolution.

One prometastatic candidate is epigenomic regulation. In particular, theinventors wished to investigate the role of large-scale epigenomicchanges during PDAC subclonal evolution and distant metastasis,especially within heterochromatin domains including large organizedheterochromatin lysine (K)-9 modified domains (LOCKs) and large DNAhypomethylated blocks. These regions could represent selectable targetsfor large-scale epigenetic reprogramming, since they occupy over half ofthe genome, partially overlap with one another, and are found in manyhuman cancers including PDAC. It was therefore hypothesized thatepigenomic dysregulation within these regions could be a major selectiveforce for tumor progression, given the lack of any consistentmetastasis-specific driver mutations.

SUMMARY OF THE INVENTION

The present invention relates to a method of identifying targets forepigenetic reprogramming comprising detecting large organizedheterochromatin lysine (K)-9 modified domains (LOCKs) and large DNAhypomethylated blocks in a sample containing DNA from a subject havingcancer. For example, the method applies to a subject that has or is atrisk of having PDAC and/or metastasis thereof. In one aspect, thedetection comprises analysis of H3K9Me2/3 and/or H4K20Me3. In anotheraspect, the detection comprises analysis of H3K27Ac and/or H3K9Ac.

In another embodiment, the invention provides for the use ofdifferentially expressed genes to identify metastatic propensity inprimary tumors, wherein the genes are selected from genes in the Tablesherein, oxidative stress genes, EMT genes, immunological response genes,DNA repair genes, glucose metabolism genes, oxPPP genes, and PGD genes.

In another embodiment, the invention provides a method for identifyingagents or compounds to affect epigenomic changes, including inhibitionof oxPPP comprising analyzing a sample from a subject before and aftercontacting with the agent or compound and determining the effect of theagent or compound on the epigenomic changes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E relate to global epigenetic reprogramming during theevolution of distant metastasis.

FIG. 1A is a series of immunohistochemical stains.

FIG. 1B is a series of immunohistochemical stains.

FIG. 1C is a series of immunohistochemical stains.

FIG. 1D is a series of immunohistochemical stains.

FIG. 1E is a series of immunohistochemical stains.

FIG. 1F is a series of western blot images.

FIG. 1G is a series of graphical representations data.

FIGS. 2A-2E relate to epigenomic reprogramming of chromatin domainsduring PDAC subclonal evolution.

FIG. 2A is a graphical representation of data.

FIG. 2B is a graphical representation of data.

FIG. 2C is a graphical representation of data.

FIG. 2D is a graphical representation of data.

FIG. 2E is a graphical representation of data.

FIGS. 3A-3G relate to reprogrammed chromatin domains encoding divergentmalignant properties.

FIG. 3A is a graphical representation of data.

FIG. 3B is a series of western blot images.

FIG. 3C is a graphical representation of data.

FIG. 3D is a series of western blot images.

FIG. 3E is a series of western blot images.

FIG. 3F is a graphical representation of data.

FIG. 3G is a series of images of tumor forming assays and relatedgraphical plots.

FIGS. 4A-4F relate to hyperactive glucose metabolism and 6PG depletionin distant metastatic subclones.

FIG. 4A is a graphical representation of data.

FIG. 4B is a graphical representation of data.

FIG. 4C is a graphical representation of data.

FIG. 4D is a schematic diagram.

FIG. 4E is a series of graphical representations of data.

FIG. 4F is a graphical representation of data.

FIGS. 5A-5D relate to PGD-dependence in distant metastatic subclones.

FIG. 5A is a series of western blot images.

FIG. 5B is a series of western blot images.

FIG. 5C is a series of images of tumor forming assays and relatedgraphical plots.

FIG. 5D is a series of images of tumor forming assays and relatedgraphical plots.

FIGS. 6A-6F relate to reversal of reprogrammed chromatin,tumorigenicity, and malignant gene expression programs by 6AN.

FIG. 6A is a series of graphical representations of data.

FIG. 6B is a series of graphical representations of data.

FIG. 6C is a series of images of tumor forming assays and relatedgraphical plots.

FIG. 6D is a series of images and related graphical plots.

FIG. 6E is a series of graphical plots.

FIG. 6F is a series of images of tumor forming assays and relatedgraphical plots.

FIGS. 7A-7C relate to reprogrammed chromatin across distant metastaticsubclones.

FIG. 7A is a series of western blot images.

FIG. 7B is a series of western blot images.

FIG. 7C is a series of western blot images.

FIGS. 8A-8E relate to specificity of reprogrammed histone modifications.

FIG. 8A is a series of immunohistochemical stains.

FIG. 8B is a graphical representation of data.

FIG. 8C is a table.

FIG. 8D is a series of western blot images.

FIG. 8E is a series of western blot images.

FIG. 9 is a series of graphical plots relating to enrichment ofheterochromatin modifications within LOCKs.

FIGS. 10A-10B relate to reprogramming of H3K9Me3 in LOCKs during PDACsubclonal evolution.

FIG. 10A is a graphical representation of data.

FIG. 10B is a graphical representation of data.

FIGS. 11A-11B relate to local reprogramming of DE gene loci withinLOCKs.

FIG. 11A is a series of graphical representations of data.

FIG. 11B is a series of graphical representations of data.

FIG. 12 is a series of graphical plots relating to enrichment ofeuchromatin modifications within ECDs.

FIGS. 13A-13E relate to reprogramming of large LOCKs during PDACevolution.

FIG. 13A is a graphical representation of data.

FIG. 13B is a graphical representation of data.

FIG. 13C is a graphical representation of data.

FIG. 13D is a graphical representation of data.

FIG. 13E is a graphical representation of data.

FIGS. 14A-14F relate to malignant heterogeneity between A38 subclones.

FIG. 14A is a graphical representation of data.

FIG. 14B is a series of graphical representations of data.

FIG. 14C is a series of immunohistochemical stains.

FIG. 14D is a series of immunohistochemical stains.

FIG. 14E is a series of western blot images.

FIG. 14F is a series of images of tumor forming assays and relatedgraphical plots.

FIGS. 15A-15C relate to rearrangements targeted to Large LOCKS and ECDs.

FIG. 15A is a series of graphical representations of data.

FIG. 15B is a series of graphical representations of data.

FIG. 15C is a series of graphical representations of data.

FIGS. 16A-16B relate to enhanced glucose metabolism with depleted 6PGlevels across distant metastases.

FIG. 16A is a series of graphical representations of data.

FIG. 16B is a series of graphical representations of data.

FIGS. 17A-17C relate to 6AN targeting of glucose metabolism and the PGDstep of the PPP.

FIG. 17A is a series of graphical representations of data.

FIG. 17B is a series of graphical representations of data.

FIG. 17C is a series of graphical representations of data.

FIGS. 18A-18C relate to 6AN selectively modulation of the reprogrammedchromatin state of distant metastatic subclones.

FIG. 18A is a series of western blot images.

FIG. 18B is a series of western blot images.

FIG. 18C is a series of western blot images.

FIGS. 19A-19D relate to 6AN regulated gene expression in LOCK-EIregions.

FIG. 19A is a series of graphical representations of data.

FIG. 19B is a series of graphical representations of data.

FIG. 19C is a series of graphical representations of data.

FIG. 19D is a series of graphical representations of data.

FIGS. 20A-20C relate to 6AN selectively blocked tumor formation indistant metastatic subclones.

FIG. 20A is a series of images of tumor forming assays and relatedgraphical plots.

FIG. 20B is a series of images of tumor forming assays and relatedgraphical plots.

FIG. 20C is a series of images of tumor forming assays and relatedgraphical plots.

FIGS. 21A-21C relates to reprogramming of the TOP2B locus in response to6AN.

FIG. 21A is a series of graphical representations of data.

FIG. 21B is a series of graphical representations of data.

FIG. 21C is a series of graphical representations of data.

FIG. 22 is a screen shot illustrating data directly linking loss oflarge-scale heterochromatic regions as described herein to increasedvariability of gene expression, allowing for increased phenotypicplasticity. An example is a gene SHC4 that is involved in ERK signalingand tumor invasion and metastasis. Its expression variabilitystatistical index measured by single cell RNA experiments is +1.12 inthe A38-5 (epigenomically altered, distant metastatic) line in thepaper, and −2.68 in the corresponding A38-41 (epigenomically stable,locally invasive) line, with a FDR p value of 0.00. FIG. 22 representsthe data showing the loss of LOCKs over the gene in 38-5.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the seminal discovery that aprometastatic candidate is epigenomic regulation. The invention is basedon discovery of the role of large-scale epigenomic changes during PDACsubclonal evolution and distant metastasis, especially withinheterochromatin domains including large organized heterochromatin lysine(K)-9 modified domains (LOCKs) and large DNA hypomethylated blocks.These regions represent selectable targets for large-scale epigeneticreprogramming, since they occupy over half of the genome, partiallyoverlap with one another, and are found in many human cancers includingPDAC. The inventors therefore hypothesized that epigenomic dysregulationwithin these regions could be a major selective force for tumorprogression, given the lack of any consistent metastasis-specific drivermutations.

Before the present systems and methods are described, it is to beunderstood that this invention is not limited to particular systems,methods, and experimental conditions described, as such systems,methods, and conditions may vary. It is also to be understood that theterminology used herein is for purposes of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only in the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described.

The present invention provides a method of identifying targets forepigenetic reprogramming comprising detecting large organizedheterochromatin lysine (K)-9 modified domains (LOCKs) and large DNAhypomethylated blocks in a sample containing DNA from a subject havingcancer. For example, the method applies to a subject that has or is atrisk of having PDAC and/or metastasis thereof. In one aspect, thedetection comprises analysis of H3K9Me2/3 and/or H4K20Me3.

As used herein, reprogramming, is intended to refer to a process thatalters or reverses the differentiation status of a somatic cell that iseither partially or terminally differentiated. Reprogramming of asomatic cell may be a partial or complete reversion of thedifferentiation status of the somatic cell. In an exemplary aspect,reprogramming is complete wherein a somatic cell is reprogrammed into aniPS cell. However, reprogramming may be partial, such as reversion intoany less differentiated state. For example, reverting a terminallydifferentiated cell into a cell of a less differentiated state, such asa multipotent cell.

As used herein, pluripotent cells include cells that have the potentialto divide in vitro for an extended period of time (greater than oneyear) and have the unique ability to differentiate into cells derivedfrom all three embryonic germ layers, namely endoderm, mesoderm andectoderm.

Somatic cells for use with the present invention may be primary cells orimmortalized cells. Such cells may be primary cells (non-immortalizedcells), such as those freshly isolated from an animal, or may be derivedfrom a cell line (immortalized cells). In an exemplary aspect, thesomatic cells are mammalian cells, such as, for example, human cells ormouse cells. They may be obtained by well-known methods, from differentorgans, such as, but not limited to skin, brain, lung, pancreas, liver,spleen, stomach, intestine, heart, reproductive organs, bladder, kidney,urethra and other urinary organs, or generally from any organ or tissuecontaining living somatic cells, or from blood cells. Mammalian somaticcells useful in the present invention include, by way of example, adultstem cells, sertoli cells, endothelial cells, granulosa epithelialcells, neurons, pancreatic islet cells, epidermal cells, epithelialcells, hepatocytes, hair follicle cells, keratinocytes, hematopoieticcells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes),erythrocytes, macrophages, monocytes, mononuclear cells, fibroblasts,cardiac muscle cells, other known muscle cells, and generally any livesomatic cells. In particular embodiments, fibroblasts are used. The termsomatic cell, as used herein, is also intended to include adult stemcells. An adult stem cell is a cell that is capable of giving rise toall cell types of a particular tissue. Exemplary adult stem cellsinclude hematopoietic stem cells, neural stem cells, and mesenchymalstem cells.

As discussed herein, alterations in methylation patterns occur duringdifferentiation or dedifferentiation of a cell which work to regulategene expression of critical factors that are ‘turned on’ or ‘turned off’at various stages of differentiation. As such, one of skill in the artwould appreciate that many types of agents are capable of altering themethylation status of one or more nucleic acid sequences of a somaticcell to induce pluripotency that may be suitable for use with thepresent invention.

An agent, as used herein, is intended to include any agent capable ofaltering the methylation status of one or more nucleic acid sequences ofa somatic cell. For example, an agent useful in any of the method of theinvention may be any type of molecule, for example, a polynucleotide, apeptide, a peptidomimetic, peptoids such as vinylogous peptoids,chemical compounds, such as organic molecules or small organicmolecules, or the like. In various aspects, the agent may be apolynucleotide, such as DNA molecule, an antisense oligonucleotide orRNA molecule, such as microRNA, dsRNA, siRNA, stRNA, and shRNA.

MicroRNA (miRNA) are single-stranded RNA molecules whose expression isknown to be regulated by methylation to play a key role in regulation ofgene expression during differentiation and dedifferentiation of cells.Thus, an agent may be one that inhibits or induces expression of miRNAor may be a mimic miRNA. As used herein, “mimic” microRNAs which areintended to mean a microRNA exogenously introduced into a cell that havethe same or substantially the same function as their endogenouscounterpart.

In various aspects of the present invention, an agent that alters themethylation status of one or more nucleic acid sequences is a nuclearreprogramming factor. Nuclear reprogramming factors may be genes thatinduce pluripotency and utilized to reprogram differentiated orsemi-differentiated cells to a phenotype that is more primitive thanthat of the initial cell, such as the phenotype of a pluripotent stemcell. Those skilled in the art would understand that such genes andagents are capable of generating a pluripotent stem cell from a somaticcell upon expression of one or more such genes having been integratedinto the genome of the somatic cell or upon contact of the somatic cellwith the agent or expression product of the gene. As used herein, a genethat induces pluripotency is intended to refer to a gene that isassociated with pluripotency and capable of generating a lessdifferentiated cell, such as a pluripotent stem cell from a somatic cellupon integration and expression of the gene. The expression of apluripotency gene is typically restricted to pluripotent stem cells andis crucial for the functional identity of pluripotent stem cells.

Several genes have been found to be associated with pluripotency andsuitable for use with the present invention as reprogramming factors.Such genes are known in the art and include, by way of example, SOXfamily genes (SOX′, SOX2, SOX3, SOX15, SOX18), KLF family genes (KLF1,KLF2, KLF4, KLF5), MYC family genes (C-MYC, L-MYC, N-MYC), SALL4, OCT4,NANOG, LIN28, STELLA, NOBOX, POU5F1 or a STAT family gene. STAT familymembers may include for example STAT1, STAT2, STAT3, STAT4, STAT5(STAT5A and STAT5B), and STAT6. While in some instances, use of only onegene to induce pluripotency may be possible, in general, expression ofmore than one gene is required to induce pluripotency. For example, two,three, four or more genes may be simultaneously integrated into thesomatic cell genome as a polycistronic construct to allow simultaneousexpression of such genes. In an exemplary aspect, four genes areutilized to induce pluripotency including OCT4, POU5F1, SOX2, KLF4 andC-MYC. Additional genes known as reprogramming factors suitable for usewith the present invention are disclosed in U.S. patent application Ser.No. 10/997,146 and U.S. patent application Ser. No. 12/289,873,incorporated herein by reference.

All of these genes commonly exist in mammals, including human, and thushomologues from any mammals may be used in the present invention, suchas genes derived from mammals including, but not limited to mouse, rat,bovine, ovine, horse, and ape. Further, in addition to wild-type geneproducts, mutant gene products including substitution, insertion, and/ordeletion of several (e.g., 1 to 10, 1 to 6, 1 to 4, 1 to 3, and 1 or 2)amino acids and having similar function to that of the wild-type geneproducts can also be used. Furthermore, the combinations of factors arenot limited to the use of wild-type genes or gene products. For example,Myc chimeras or other Myc variants can be used instead of wild-type Myc.

The present invention is not limited to any particular combination ofnuclear reprogramming factors. As discussed herein a nuclearreprogramming factor may comprise one or more gene products. The nuclearreprogramming factor may also comprise a combination of gene products asdiscussed herein. Each nuclear reprogramming factor may be used alone orin combination with other nuclear reprogramming factors as disclosedherein. Further, nuclear reprogramming factors of the present inventioncan be identified by screening methods, for example, as discussed inU.S. patent application Ser. No. 10/997,146, incorporated herein byreference. Additionally, the nuclear reprogramming factor of the presentinvention may contain one or more factors relating to differentiation,development, proliferation or the like and factors having otherphysiological activities, as well as other gene products which canfunction as a nuclear reprogramming factor.

The nuclear reprogramming factor may include a protein or peptide. Theprotein may be produced from a gene as discussed herein, oralternatively, in the form of a fusion gene product of the protein withanother protein, peptide or the like. The protein or peptide may be afluorescent protein and/or a fusion protein. For example, a fusionprotein with green fluorescence protein (GFP) or a fusion gene productwith a peptide such as a histidine tag can also be used. Further, bypreparing and using a fusion protein with the TAT peptide derived fromthe virus HIV, intracellular uptake of the nuclear reprogramming factorthrough cell membranes can be promoted, thereby enabling induction ofreprogramming only by adding the fusion protein to a medium thusavoiding complicated operations such as gene transduction. Sincepreparation methods of such fusion gene products are well known to thoseskilled in the art, skilled artisans can easily design and prepare anappropriate fusion gene product depending on the purpose.

In certain embodiments, the agent alters the methylation status of oneor more nucleic acid sequences, such as any gene listed in a Table setforth herein.

Expression profiling of reprogrammed somatic cells to assess theirpluripotency characteristics may also be conducted. Expression ofindividual genes associated with pluripotency may also be examined.Additionally, expression of embryonic stem cell surface markers may beanalyzed. As used herein, “expression” refers to the production of amaterial or substance as well as the level or amount of production of amaterial or substance. Thus, determining the expression of a specificmarker refers to detecting either the relative or absolute amount of themarker that is expressed or simply detecting the presence or absence ofthe marker. As used herein, “marker” refers to any molecule that can beobserved or detected. For example, a marker can include, but is notlimited to, a nucleic acid, such as a transcript of a specific gene, apolypeptide product of a gene, a non-gene product polypeptide, aglycoprotein, a carbohydrate, a glycolipd, a lipid, a lipoprotein or asmall molecule.

Detection and analysis of a variety of genes known in the art to beassociated with pluripotent stem cells may include analysis of genessuch as, but not limited to OCT4, NANOG, SALL4, SSEA-1, SSEA-3, SSEA-4,TRA-1-60, TRA-1-81, or a combination thereof iPS cells may express anynumber of pluripotent cell markers, including: alkaline phosphatase(AP); ABCG2; stage specific embryonic antigen-1 (SSEA-1); SSEA-3;SSEA-4; TRA-1-60; TRA-1-81; Tra-2-49/6E; ERas/ECATS, E-cadherin;β-III-tubulin; γ-smooth muscle actin (γ-SMA); fibroblast growth factor 4(Fgf4), Cripto, Dax1; zinc finger protein 296 (Zfp296);N-acetyltransferase-1 (Nat1); ES cell associated transcript 1 (ECAT1);ESG1/DPPA5/ECAT2; ECAT3; ECAT6; ECAT7; ECAT8; ECAT9; ECAT10; ECAT15-1;ECAT15-2; Fth117; Sa114; undifferentiated embryonic cell transcriptionfactor (Utf1); Rex1; p53; G3PDH; telomerase, including TERT; silent Xchromosome genes; Dnmt3a; Dnmt3b; TRIM28; F-box containing protein(Fbx15); Nanog/ECAT4; Oct3/4; Sox2; Klf4; c-Myc; Esrrb; TDGF1; GABRB3;Zfp42, FoxD3; GDF3; CYP25A1; developmental pluripotency-associated 2(DPPA2); T-cell lymphoma breakpoint 1 (Tcl1); DPPA3/Stella; DPPA4; aswell as other general markers for pluripotency, for example any genesused during induction to reprogram the cell. iPS cells can also becharacterized by the down-regulation of markers characteristic of thedifferentiated cell from which the iPS cell is induced.

As used herein, “differentiation” refers to a change that occurs incells to cause those cells to assume certain specialized functions andto lose the ability to change into certain other specialized functionalunits. Cells capable of differentiation may be any of totipotent,pluripotent or multipotent cells. Differentiation may be partial orcomplete with respect to mature adult cells.

“Differentiated cell” refers to a non-embryonic, non-parthenogenetic ornon-pluripotent cell that possesses a particular differentiated, i.e.,non-embryonic, state. The three earliest differentiated cell types areendoderm, mesoderm, and ectoderm.

Pluripotency can also be confirmed by injecting the cells into asuitable animal, e.g., a SCID mouse, and observing the production ofdifferentiated cells and tissues. Still another method of confirmingpluripotency is using the subject pluripotent cells to generate chimericanimals and observing the contribution of the introduced cells todifferent cell types. Methods for producing chimeric animals are wellknown in the art and are described in U.S. Pat. No. 6,642,433,incorporated by reference herein.

Yet another method of confirming pluripotency is to observe celldifferentiation into embryoid bodies and other differentiated cell typeswhen cultured under conditions that favor differentiation (e.g., removalof fibroblast feeder layers).

In various aspects of the invention, methylation status is converted toan M value. As used herein an M value, can be a log ratio of intensitiesfrom total (Cy3) and McrBC-fractionated DNA (Cy5): positive and negativeM values are quantitatively associated with methylated and unmethylatedsites, respectively.

In various aspects of the invention large hypomethylated blocks areidentified Hypomethylation is present when there is a measurabledecrease in methylation. In some embodiments, a DNA block ishypomethylated when less than 50% of the methylation sites analyzed arenot methylated. DNA block. Methods for determining methylation statesare provided herein and are known in the art. In some embodimentsmethylation status is converted to an M value. As used herein an Mvalue, can be a log ratio of intensities from total (Cy3) andMcrBC-fractionated DNA (Cy5): positive and negative M values arequantitatively associated with methylated and unmethylated sites,respectively. M values are calculated as described in the Examples. Insome embodiments, M values which range from −0.5 to 0.5 representunmethylated sites as defined by the control probes, and values from 0.5to 1.5 represent baseline levels of methylation.

Numerous methods for analyzing methylation status of a gene are known inthe art and can be used in the methods of the present invention toidentify either hypomethylation or hypermethylation of the one or moreDMRs. In various embodiments, the determining of methylation status inthe methods of the invention is performed by one or more techniquesselected from the group consisting of a nucleic acid amplification,polymerase chain reaction (PCR), methylation specific PCR, bisulfitepyrosequencing, single-strand conformation polymorphism (SSCP) analysis,restriction analysis, microarray technology, and proteomics. Asillustrated in the Examples herein, analysis of methylation can beperformed by bisulfite genomic sequencing. Bisulfite treatment modifiesDNA converting unmethylated, but not methylated, cytosines to uracil.Bisulfite treatment can be carried out using the METHYLEASY bisulfitemodification kit (Human Genetic Signatures).

In some embodiments, bisulfite pyrosequencing, which is asequencing-based analysis of DNA methylation that quantitativelymeasures multiple, consecutive CpG sites individually with high accuracyand reproducibility, may be used.

It will be recognized that depending on the site bound by the primer andthe direction of extension from a primer, that the primers listed abovecan be used in different pairs. Furthermore, it will be recognized thatadditional primers can be identified within the hypomethylated blocks,especially primers that allow analysis of the same methylation sites asthose analyzed with primers that correspond to the primers disclosedherein.

Altered methylation can be identified by identifying a detectabledifference in methylation. For example, hypomethylation can bedetermined by identifying whether after bisulfite treatment a uracil ora cytosine is present a particular location. If uracil is present afterbisulfite treatment, then the residue is unmethylated. Hypomethylationis present when there is a measurable decrease in methylation.

In an alternative embodiment, the method for analyzing methylation of ahypomethylated block can include amplification using a primer pairspecific for methylated residues within a DMR. In these embodiments,selective hybridization or binding of at least one of the primers isdependent on the methylation state of the target DNA sequence (Herman etal., Proc. Natl. Acad. Sci. USA, 93:9821 (1996)). For example, theamplification reaction can be preceded by bisulfite treatment, and theprimers can selectively hybridize to target sequences in a manner thatis dependent on bisulfite treatment. For example, one primer canselectively bind to a target sequence only when one or more base of thetarget sequence is altered by bisulfite treatment, thereby beingspecific for a methylated target sequence.

Other methods are known in the art for determining methylation status ofa hypomethylated block, including, but not limited to, array-basedmethylation analysis and Southern blot analysis.

Methods using an amplification reaction, for example methods above fordetecting hypomethylation or hypermethylation of one or morehypomethylated blocks, can utilize a real-time detection amplificationprocedure. For example, the method can utilize molecular beacontechnology (Tyagi et al., Nature Biotechnology, 14: 303 (1996)) orTaqman™ technology (Holland et al., Proc. Natl. Acad. Sci. USA, 88:7276(1991)).

Also methyl light (Trinh et al., Methods 25(4):456-62 (2001),incorporated herein in its entirety by reference), Methyl Heavy(Epigenomics, Berlin, Germany), or SNuPE (single nucleotide primerextension) (see e.g., Watson et al., Genet Res. 75(3):269-74 (2000)) Canbe used in the methods of the present invention related to identifyingaltered methylation of DMRs.

As used herein, the term “selective hybridization” or “selectivelyhybridize” refers to hybridization under moderately stringent or highlystringent physiological conditions, which can distinguish relatednucleotide sequences from unrelated nucleotide sequences.

As known in the art, in nucleic acid hybridization reactions, theconditions used to achieve a particular level of stringency will vary,depending on the nature of the nucleic acids being hybridized. Forexample, the length, degree of complementarity, nucleotide sequencecomposition (for example, relative GC:AT content), and nucleic acidtype, for example, whether the oligonucleotide or the target nucleicacid sequence is DNA or RNA, can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter. Methods forselecting appropriate stringency conditions can be determinedempirically or estimated using various formulas, and are well known inthe art (see, e.g., Sambrook et al., supra, 1989).

An example of progressively higher stringency conditions is as follows:2×SSC/0.1% SDS at about room temperature (hybridization conditions);0.2×SSC/0.1% SDS at about room temperature (low stringency conditions);0.2×SSC/0.1% SDS at about 42° C. (moderate stringency conditions); and0.1×SSC at about 68° C. (high stringency conditions). Washing can becarried out using only one of these conditions, for example, highstringency conditions, or each of the conditions can be used, forexample, for 10 to 15 minutes each, in the order listed above, repeatingany or all of the steps listed.

The degree of methylation in the DNA associated with the DMRs beingassessed, may be measured by fluorescent in situ hybridization (FISH) bymeans of probes which identify and differentiate between genomic DNAs,associated with the DMRs being assessed, which exhibit different degreesof DNA methylation. FISH is described, for example, in de Capoa et al.(Cytometry. 31:85-92, 1998) which is incorporated herein by reference.In this case, the biological sample will typically be any which containssufficient whole cells or nuclei to perform short term culture. Usually,the sample will be a sample that contains 10 to 10,000, or, for example,100 to 10,000, whole cells.

Additionally, as mentioned above, methyl light, methyl heavy, andarray-based methylation analysis can be performed, by using bisulfatetreated DNA that is then PCR-amplified, against microarrays ofoligonucleotide target sequences with the various forms corresponding tounmethylated and methylated DNA.

The term “nucleic acid molecule” is used broadly herein to mean asequence of deoxyribonucleotides or ribonucleotides that are linkedtogether by a phosphodiester bond. As such, the term “nucleic acidmolecule” is meant to include DNA and RNA, which can be single strandedor double stranded, as well as DNA/RNA hybrids. Furthermore, the term“nucleic acid molecule” as used herein includes naturally occurringnucleic acid molecules, which can be isolated from a cell, as well assynthetic molecules, which can be prepared, for example, by methods ofchemical synthesis or by enzymatic methods such as by the polymerasechain reaction (PCR), and, in various embodiments, can containnucleotide analogs or a backbone bond other than a phosphodiester bond.

The terms “polynucleotide” and “oligonucleotide” also are used herein torefer to nucleic acid molecules. Although no specific distinction fromeach other or from “nucleic acid molecule” is intended by the use ofthese terms, the term “polynucleotide” is used generally in reference toa nucleic acid molecule that encodes a polypeptide, or a peptide portionthereof, whereas the term “oligonucleotide” is used generally inreference to a nucleotide sequence useful as a probe, a PCR primer, anantisense molecule, or the like. Of course, it will be recognized thatan “oligonucleotide” also can encode a peptide. As such, the differentterms are used primarily for convenience of discussion.

A polynucleotide or oligonucleotide comprising naturally occurringnucleotides and phosphodiester bonds can be chemically synthesized orcan be produced using recombinant DNA methods, using an appropriatepolynucleotide as a template. In comparison, a polynucleotide comprisingnucleotide analogs or covalent bonds other than phosphodiester bondsgenerally will be chemically synthesized, although an enzyme such as T7polymerase can incorporate certain types of nucleotide analogs into apolynucleotide and, therefore, can be used to produce such apolynucleotide recombinantly from an appropriate template.

In another aspect, the present invention includes kits that are usefulfor carrying out the methods of the present invention. The componentscontained in the kit depend on a number of factors, including: theparticular analytical technique used to detect methylation or measurethe degree of methylation or a change in methylation, and the one ormore hypomethylated blocks being assayed for.

Accordingly, the present invention provides a kit for determining amethylation status of one or more hypomethylated blocks of theinvention.

To examine DNA methylation (DNAm) on a genome-wide scale, comprehensivehigh-throughput array-based relative methylation (CHARM) analysis, whichis a microarray-based method agnostic to preconceptions about DNAm,including location relative to genes and CpG content was carried out.The resulting quantitative measurements of DNAm, denoted with M, are logratios of intensities from total (Cy3) and McrBC-fractionated DNA (Cy5):positive and negative M values are quantitatively associated withmethylated and unmethylated sites, respectively. For each sample, ˜4.6million CpG sites across the genome of iPS cells, parental somatic cellsand ES cells were analyzed using a custom-designed NimbleGen HD2microarray, including all of the classically defined CpG islands as wellas all nonrepetitive lower CpG density genomic regions of the genome.4,500 control probes were included to standardize these M values so thatunmethylated regions were associated, on average, with values of 0.CHARM is 100% specific at 90% sensitivity for known methylation marksidentified by other methods (for example, in promoters) and includes theapproximately half of the genome not identified by conventional regionpreselection. The CHARM results were also extensively corroborated byquantitative bisulfate pyrosequencing analysis.

In one aspect of the invention, methylation density is determined for aregion of nucleic acid. Density may be used as an indication of ahypomethylated block region of DNA, for example. A density of about 0.2to 0.7, about 0.3 to 0.7, 0.3 to 0.6 or 0.3 to 0.4, or 0.3, may beindicative of a hypomethylated block (the calculated DNA methylationdensity is the number of methylated CpGs divided by the total number ofCpGs sequenced for each sample). Methods for determining methylationdensity are well known in the art. For example, a method for determiningmethylation density of target CpG islands has been established by Luo etal. Analytical Biochemistry, Vol. 387:2 2009, pp. 143-149. In themethod, DNA microarray was prepared by spotting a set of PCR productsamplified from bisulfite-converted sample DNAs. This method not onlyallows the quantitative analysis of regional methylation density of aset of given genes but also could provide information of methylationdensity for a large amount of clinical samples as well as use in themethods of the invention regarding iPS cell generation and detection.Other methods are well known in the art (e.g., Holemon et al.,BioTechniques, 43:5, 2007, pp. 683-693).

The present invention is described partly in terms of functionalcomponents and various processing steps. Such functional components andprocessing steps may be realized by any number of components, operationsand techniques configured to perform the specified functions and achievethe various results. For example, the present invention may employvarious biological samples, biomarkers, elements, materials, computers,data sources, storage systems and media, information gatheringtechniques and processes, data processing criteria, statisticalanalyses, regression analyses and the like, which may carry out avariety of functions. In addition, although the invention is describedin the medical diagnosis context, the present invention may be practicedin conjunction with any number of applications, environments and dataanalyses; the systems described herein are merely exemplary applicationsfor the invention.

Methods for analysis according to various aspects of the presentinvention may be implemented in any suitable manner, for example using acomputer program operating on the computer system. An exemplary analysissystem, according to various aspects of the present invention, may beimplemented in conjunction with a computer system, for example aconventional computer system comprising a processor and a random accessmemory, such as a remotely-accessible application server, networkserver, personal computer or workstation. The computer system alsosuitably includes additional memory devices or information storagesystems, such as a mass storage system and a user interface, for examplea conventional monitor, keyboard and tracking device. The computersystem may, however, comprise any suitable computer system andassociated equipment and may be configured in any suitable manner. Inone embodiment, the computer system comprises a stand-alone system. Inanother embodiment, the computer system is part of a network ofcomputers including a server and a database.

The software required for receiving, processing, and analyzing biomarkerinformation may be implemented in a single device or implemented in aplurality of devices. The software may be accessible via a network suchthat storage and processing of information takes place remotely withrespect to users. The analysis system according to various aspects ofthe present invention and its various elements provide functions andoperations to facilitate biomarker analysis, such as data gathering,processing, analysis, reporting and/or diagnosis. The present analysissystem maintains information relating to methylation and samples andfacilitates analysis and/or diagnosis. For example, in the presentembodiment, the computer system executes the computer program, which mayreceive, store, search, analyze, and report information relating to theepigenome. The computer program may comprise multiple modules performingvarious functions or operations, such as a processing module forprocessing raw data and generating supplemental data and an analysismodule for analyzing raw data and supplemental data to generate adisease status model and/or diagnosis information.

The procedures performed by the analysis system may comprise anysuitable processes to facilitate analysis and/or disease diagnosis. Inone embodiment, the analysis system is configured to establish a diseasestatus model and/or determine disease status in a patient. Determiningor identifying disease status may comprise generating any usefulinformation regarding the condition of the patient relative to thedisease, such as performing a diagnosis, providing information helpfulto a diagnosis, assessing the stage or progress of a disease,identifying a condition that may indicate a susceptibility to thedisease, identify whether further tests may be recommended, predictingand/or assessing the efficacy of one or more treatment programs, orotherwise assessing the disease status, likelihood of disease, or otherhealth aspect of the patient.

The analysis system may also provide various additional modules and/orindividual functions. For example, the analysis system may also includea reporting function, for example to provide information relating to theprocessing and analysis functions. The analysis system may also providevarious administrative and management functions, such as controllingaccess and performing other administrative functions.

The analysis system suitably generates a disease status model and/orprovides a diagnosis for a patient based on raw biomarker data and/oradditional subject data relating to the subjects. The data may beacquired from any suitable biological samples.

The following examples are provided to further illustrate the advantagesand features of the present invention but are not intended to limit thescope of the invention. While they are typical of those that might beused, other procedures, methodologies, or techniques known to thoseskilled in the art may alternatively be used.

Example 1 Large-Scale Epigenomic Reprogramming Links Anabolic GlucoseMetabolism to Distant Metastasis During the Evolution of PancreaticCancer Progression Background and Hypothesis

As discussed previously, one prometastatic candidate is epigenomicregulation. In particular, we wished to investigate the role oflarge-scale epigenomic changes during PDAC subclonal evolution anddistant metastasis, especially within heterochromatin domains includinglarge organized heterochromatin lysine (K)-9 modified domains (LOCKs)and large DNA hypomethylated blocks. These regions could representselectable targets for large-scale epigenetic reprogramming, since theyoccupy over half of the genome, partially overlap with one another, andare found in many human cancers including PDAC. The inventors thereforehypothesized that epigenomic dysregulation within these regions could bea major selective force for tumor progression, given the lack of anyconsistent metastasis-specific driver mutations.

Results Reprogramming of Global Epigenetic State During the Evolution ofDistant Metastasis

To test this hypothesis, we first determined whether large-scale changesin epigenetic modifications could be detected during PDAC evolution inpatient samples in vivo. We previously collected matched primary andmetastatic PDAC lesions from individual patients by rapid autopsy andreported the genetic progression of subclonal evolution by whole exomeand sanger sequencing for mutations, paired-end sequencing forrearrangements, and whole genome sequencing in a subset of thesesamples. These samples represent a unique resource especially suited tostudy tumor evolution, since they were collected from matched primaryand metastatic tumors from the same patient(s), each has been deepsequenced, individual subclones have been identified, and nometastasis-specific driver mutations are present. From these patients,we selected a large panel of diverse PDAC samples to test for globalepigenomic reprogramming during subclonal evolution. As summarized inTable 1, these samples were chosen because they represented thediversity of PDAC evolution (different regions of primary tumor pairedto peritoneal and distant metastases), each sample represented asequence-verified (sub)clonal population, patients were both treated anduntreated, driver mutations were shared by all subclones in each patientin the absence of metastasis-specific drivers, formalin-fixed tissue wasavailable for immunoassays, frozen tissue was available for whole-genomebisulfate sequencing, and cell lines were available for all otherexperiments.

TABLE 1 PDAC sample characteristics with LOCK epigenetic changesAssays^(b) Sample Driver Genes IHC, Western Results^(b) (Clonal Disease(in primary Blots, ChIP- LOCK Methylation Relation^(a)) Present. Sourceand mets) Chemo seq, WGBS Change A124PrF Local- Tissue: KRAS UntreatedK9Me2/3 IHC: Diffusely Positive (Founder regional Primary CDKN2A Clone)Tumor SMAD4 ATM A124PrS Local- Tissue: KRAS Untreated K9Me2/3 IHC:Diffusely Positive (Subclone) regional Primary CDKN2A WGBS: High (75%,control) Tumor SMAD4 ATM A124Per Local- Tissue: KRAS Untreated K9Me2/3IHC: Diffusely Positive (Metastasis) regional Peritoneum CDKN2A WGBS:High (74%) SMAD4 ATM A141PrF Local- Tissue: KRAS Untreated K9Me2/3 IHC:Diffusely Positive (Founder regional Primary CDKN2A Clone) Tumor TP53MLL3 ARID1B A141PrS Local- Tissue: KRAS Untreated K9Me2/3 IHC: DiffuselyPositive (Subclone) regional Primary CDKN2A Tumor TP53 MLL3 ARID1BA141Per Local- Tissue: KRAS Untreated K9Me2/3 IHC: Diffusely Positive(Metastasis) regional Peritoneum CDKN2A TP53 MLL3 ARID1B A125PrF DistantTissue: KRAS Untreated K9Me2/3 IHC: Diffusely Positive (Foundermetastases Primary CDKN2A WGBS: High (74%) Clone) Tumor TP53 ARID1AA125PrS Distant Tissue: KRAS Untreated K9Me2/3 IHC: Positive + Negative(Subclone) metastases Primary CDKN2A WGBS: Reduced (51%) Tumor TP53ARID1A A125Lvl Distant Tissue: KRAS Untreated K9Me2/3 IHC: DiffuselyNegative (Metastasis) metastases Liver CDKN2A WGBS: Reduced (57%) TP53ARID1A A125Lv2 Distant Tissue: KRAS Untreated K9Me2/3 IHC: DiffuselyNegative (Metastasis) metastases Liver CDKN2A WGBS: Reduced (59%) TP53ARID1A A132PrF Distant Tissue: KRAS Untreated K9Me2/3 IHC: DiffuselyPositive (Founder metastases Primary CDKN2A Clone) Tumor TP53 ATMA132PrS Distant Tissue: KRAS Untreated K9Me2/3 IHC: Positive + Negative(Subclone) metastases Primary CDKN2A Tumor TP53 ATM A132Lv DistantTissue: KRAS Untreated K9Me2/3 IHC: Positive + Negative (Metastasis)metastases Liver CDKN2A TP53 ATM A38PrF Local- Tissue: KRAS Gem. K9Me2/3IHC: Diffusely Positive (Founder regional + Primary TP53 Bev. Clone)Distant Tumor SMAD4 metastases A38PrS1 Local- Tissue: KRAS Gem. K9Me2/3IHC: Diffusely Positive (Peritoneal regional + Primary TP53 Bev.Precursor Distant Tumor SMAD4 Subclone) metastases A38PrS2 Local-Tissue: KRAS Gem. K9Me2/3 IHC: Positive + Negative (Liver/Lungregional + Primary TP53 Bev. Precursor Distant Tumor SMAD4 Subclone)metastases SMARCA2^(c) A38Lg1 Local- Tissue: KRAS Gem. K9Me2/3 IHC:Diffusely Negative (Metastasis) regional + Lung TP53 Bev. Distant SMAD4metastases A38Per Local- Cell Line: KRAS Gem. K9Me2 High (100%, cont.)(Metastasis) regional + Peritoneum TP53 Bev. Western Blot: Distant SMAD4K9Me2 High (100%, cont.) metastases ChIP-seq: WGBS: High (79%, cont.)AsPC1^(d) Local- Cell Line: KRAS Gem. K9Me2 High (102%) (N/A) regionalAscites CDKN2A Western Blot: TP53 SMAD4 HPAFII^(d) Local- Cell Line:KRAS Gem. K9Me2 High (94%) (N/A⁾ regional Ascites TP53 Western Blot:Capan2^(d) Local- Cell Line: KRAS Gem. K9Me2 High (93%) (N/A) regionalPrimary CDKN2A Western Blot: Tumor TP53 A2Lg Local- Cell Line: KRASTaxoprex. K9Me2 Reduced (40%) (Metastasis) regional + Lung TP53 Gem.Western Blot: Distant metastases A2Lv Local- Cell Line: KRAS Taxoprex.K9Me2 Reduced (50%) (Metastasis) regional + Liver TP53 Gem. WesternBlot: Distant metastases A6Lv Local- Cell Line: KRAS Gem. K9Me2 Reduced(47%) (Metastasis) regional + Liver TP53 Trox. Western Blot: DistantMLL3 metastases A10Lv Distant Cell Line: KRAS Untreated K9Me2 Reduced(61%) (Metastasis) metastases Liver TP53 Western Blot: MLL3 A13Pr1Distant Cell Line: KRAS Untreated K9Me2 Reduced (64%) (Subclone)metastases Primary CDKN2A Western Blot: Tumor MYC K9Me2 Reduced (25%)TP53 ChIP-seq: WGBS: Reduced (72%) A13Pr2 Distant Cell Line: KRASUntreated K9Me2 Reduced (51%) (Subclone) metastases Primary CDKN2AWestern Blot: Tumor MYC K9Me2 Reduced (81%) TP53 ChIP-seq: WGBS: Reduced(73%) A13Lg Distant Cell Line: KRAS Untreated K9Me2/3 Reduced (53%)(Metastasis) metastases Lung CDKN2A Western Blot: MYC K9Me2 Reduced(86%) TP53 ChIP-seq: WGBS: Reduced (71%) A32O Local- Cell Line: KRAS5-FU K9Me2 Reduced (58%) (Metastasis) regional + Omentum^(e) TP53Western Blot: Distant metastases A38Lv Local- Cell Line: KRAS Gem. K9Me2Reduced (47%) (Metastasis) regional + Liver TP53 Bev. Western Blot:Distant SMAD4 WGBS: Reduced (67%) metastases A38Lg Local- Cell Line:KRAS Gem. K9Me2 Reduced (58%) (Metastasis) regional + Lung TP53 Bev.Western Blot: Distant SMAD4 K9Me2 Reduced (48%) metastases ChIP-seq:WGBS: Reduced (72%) Table 1: Summary of H3K9 and DNA methylation changesacross tissue and cell line samples. Samples from patients withlocal-regional spread (peritoneal/ascites) showed relatively high globalH3K9/DNA methylation as indicated by multiple assays (right twocolumns), while samples from patients with distant metastases showedreduced methylation across all assays, which initiated in primary tumorsas indicated. Abbreviations: Gem. (Gemcitabine), Bev. (Bevacizumab),Taxoprex. (Taxoprexin), Trox. (Troxacitabine). Superscript Notes^(a)Clonal origins represent phylogenetic estimates from previouslypublished (ref. 1) and other unpublished (text footnote 1) whole-genomesequencing data. ^(b)Western blot data reflect densitometry percentagesof H3K9Me2 signals relative to A38Per controls (cont.). Western blotsare shown in Supplementary FIG. 1 and the absolute densitometry valuesare shown in FIG. 1g with p-values included in the figure legend.ChIP-seq data reflect percent of LOCK Mb with reduced H3K9Me2 relativeto A38Per controls (cont.), as detailed with Mb and RPKM values inSupplementary Data File 2. WGBS data reflect percent of DNA methylationwithin LOCKs relative to A124Pr controls (tissues) and A38Per controls(cell lines), as detailed in Supplementary FIGS. 1 and 2. ^(c)Thismetastasis from a chemotherapy-treated patient had a missense mutationin SMARCA2 of unclear significance. ^(d)These cell lines were not fromthe rapid autopsy cohort and rely on previously published genotypingdata which may underestimate the driver mutations. ^(e)The A32O cellline was isolated from an omental mass lesion in a patient with veryaggressive disease including widespread lung metastases, and showedfindings similar to the other distant (lung/liver) metastatic subclones.

We began our analysis with the formalin-fixed tissue samples (totaling16 uniquely matched, sequence-verified tumor sections, Table 1). Weperformed immunostains against heterochromatin modifications (e.g.,H3K9Me2/3) in order to detect global changes from heterochromatindomains (including LOCKs as defined in Wen et al. (Large histone H3lysine 9 dimethylated chromatin blocks distinguish differentiated fromembryonic stem cells. Nat Genet 41, 246-50 (2009)), and McDonald et al.(Genome-scale epigenetic reprogramming during epithelial-to-mesenchymaltransition. Nat Struct Mol Biol 18, 867-74 (2011))) that might beselectable targets during subclonal evolution. Immunostains revealeddiffusely positive (>80%) H3K9Me2/3 staining of PDAC cell nuclei acrossboth primary tumor and peritoneal metastatic subclones from patients whopresented with peritoneal carcinomatosis (FIGS. 1A-1B and Table 1). Incontrast, samples from patients who presented with distant metastaticdisease displayed progressive loss of H3K9Me2/3 during subclonalevolution. This manifested as heterogeneous (mixtures ofpositive+negative PDAC nuclei) staining in primary tumors followed byeither diffusely negative (<20%) staining or retention of heterogeneousstaining in the paired metastases (FIGS. 1C-1D and Table 1). We alsoobserved similar results during subclonal evolution from a patient forwhich sequence-verified primary tumor subclones that seeded bothperitoneal and distant metastases were available. The peritonealprecursor retained diffusely strong staining of heterochromatinmodifications as seen in the clone that founded the neoplasm (FIG. 1E,top two panels). In contrast, cell-to-cell heterogeneity of stainingpatterns emerged in the primary tumor subclone that seeded distantmetastasis, followed by diffuse loss of staining at the distantmetastatic site (FIG. 1E, bottom two panels). The collective findingsacross samples from patients with distant metastatic disease thussuggested that reprogramming initiated during subclonal evolution in theprimary tumor, and that these changes were inherited or even accentuatedin subclones that formed tumors at the distant metastatic sitesthemselves.

To expand our analysis to more patient samples and test the generalityof our findings, we employed twelve low-passaged cell lines collectedfrom eight patients (Table 1), including a subset that corresponded tothe patient tissues above. Cell lines were isolated from nine distantmetastatic subclones, a peritoneal metastasis with paired liver and lungmetastases that corresponded to the patient presented in FIG. 1E, andtwo (non-founder) primary tumor subclones matched to a lung metastasiscollected from the same patient. Importantly, six of the nine distantmetastatic cell lines were previously whole exome sequenced, andmutations present in the cell lines were also present in thecorresponding patient tissues as detected by Sanger sequencing. Becauserapid autopsy cell lines were largely isolated from patients whopresented with distant metastases, additional PDAC samples from othersources of regional disease were also included: malignant ascites fluidfrom two patients with peritoneal carcinomatosis (AsPC1, HPAFII), and aprimary tumor from a long-term survivor without distant metastases(Capan2).

We then examined whether global changes in chromatin as seen in thepatient tissues were maintained in cell lines, which could reflectgenome-wide reprogramming events. We began by performing western blotsfor eight histone modifications with well-understood functions.Comparison of local-regional PDAC samples (A38Per, AsPC1, HPAFII,Capan2) by western blots showed minimal or non-recurrent global changesacross all histone modifications tested (FIG. 7A), similar to theevolution of peritoneal carcinomatosis observed in patient tissues. Incontrast, distant metastases displayed striking reprogramming ofmethylation and acetylation that was targeted to specific histoneresidues (FIG. 7B), including between the paired peritoneal (A38Per) anddistant metastatic (A38Lv, A38Lg) subclones that corresponded to thepatient presented in FIG. 1E (FIG. 1F). This manifested as recurrentreductions in H3K9Me2/3 and H4K20Me3 that was coupled tohyper-acetylation of H3K9Ac and H3K27Ac in distant metastases(summarized in FIG. 1G and Table 1, shown in FIG. 7B). H3K9 methylationis critical for encoding heterochromatic epigenetic states over largegenomic regions including LOCKs, and H3K27Ac encodes gene regulatoryelements. Reprogramming appeared specific, as we observed noconsistent/recurrent changes in H3K27Me3 or H3K36Me3 across samples(FIG. 1G, FIGS. 7A-7C) and the reprogrammed modifications themselveswere not dependent on proliferation rates and could not be induced byPDAC chemotherapy (FIGS. 8A-8E). Finally, western blots on cell linesisolated from matched primary tumor (A13Pr1, A13Pr2) and distantmetastatic (A13Lg) subclones collected from a patient who presented withwidespread distant metastases in the absence of regional (peritoneal)spread also showed reductions in H3K9Me3 and H4K20Me3 between theprimary tumor subclones, which was retained in the distant metastasis(FIG. 7C), further suggesting that reprogramming initiated in theprimary tumor during the evolution of distant metastasis. Thus, in vivotissue and in vitro cell culture findings across the collective 30patient samples strongly suggested that global epigenetic state wasreprogrammed during the evolution of distant metastasis.

The Epigenomic Landscape of PDAC Subclonal Evolution

We next wished to map the locations of reprogrammed chromatinmodifications across the PDAC genome. To this end, we comprehensivelymapped the epigenetic landscape of PDAC evolution with chromatinimmunoprecipitation followed by high-throughput sequencing (ChIP-seq)for histone modifications with well-understood functions(heterochromatin: H3K9Me2, H3K9Me3, H3K27Me3; euchromatin: H3K27Ac,H3K36Me3). To capture the diversity of subclonal evolution and malignantprogression, ChIP-seq was performed on sequence-verified cell linesisolated from matched subclones, including a peritoneal metastasis(A38Per) matched to a lung metastasis (A38Lg) from the same patient, andtwo primary tumor subclones (A13Pr1, A13Pr2) that were also matched to alung metastasis (A13Lg) from the same patient. For each patient, allsubclones shared identical driver gene mutations without acquisition ofnew metastasis-specific drivers (Table 1). We also performed RNA-seq inparallel to identify matched gene expression changes. Finally, wecomplemented these datasets with whole genome bisulfate sequencing(WGBS) across these cell lines and frozen tumor tissues thatcorresponded to a subset of the formalin-fixed tumor sections presentedin FIGS. 1A-1G. In all, we generated 183 datasets with 19.3×10 9uniquely aligned sequencing reads, including >15.0×10⁶ (median: 32.3×106) uniquely aligned reads for each ChIP experiment as recommended byENCODE guidelines (Supplementary Data 1). Experiments were performed asbiological replicates, with good correlation between replicates (mediancorrelation coefficient: 0.956; range: 0.746-0.997, Supplementary Data1). To our knowledge, this represents the first comprehensivegenome-wide analysis of epigenetic reprogramming during the evolutionaryprogression of a human cancer.

Because global chromatin modifications were stably inherited during theevolution of peritoneal carcinomatosis whereas reprogramming emergedduring distant metastasis, we began by comparing the peritoneal subcloneagainst the distant metastatic subclones and their matched primary tumorsubclones. This analysis revealed a striking and unexpected degree ofgenome-wide epigenetic reprogramming that was targeted to thousands ofchromatin domains covering >95% of the PDAC genome. These includedheterochromatin regions corresponding to large organized chromatinlysine (K)-modified domains (LOCKs) that occupied approximately half thegenome, gene-rich euchromatin domains (ECDs), and a smaller subset ofvery large LOCK domains that were uniquely reprogrammed compared toother heterochromatin regions. The collective domain characteristics aredetailed in Supplementary Data 2, and comprehensive statistical analysesacross experiments are presented in Supplementary Data 3.

We first analyzed heterochromatin domains defined by strong, broadenrichments of H3K9Me2 and H3K27Me3 with depletion of euchromatinmodifications (FIG. 2A, FIG. 9 , and Supplementary methods). Thousandsof heterochromatin domains were detected in each sample (range:2,008-3,166) and were organized into large block-like segments (medianlengths: 232 Kb-311 Kb) that occupied more than half of the genome ineach subclone (average: 61.7% of the genome, range: 54.1-71.7%,Supplementary Data 2). The domain calls were robust as determined bysensitivity analyses across multiple thresholds (Supplementary Data 3),and the called heterochromatin regions themselves overlappedsignificantly with previously reported LOCK heterochromatin domains(avg: 76.7+/−16.9% overlap; p<0.01 by permutation testing), suggestingthat heterochromatin largely corresponded to LOCKs. Similar toimmunostain (FIGS. 1A-1D) and western blot data (FIG. 1E-1F), wedetected strong H3K9Me2 enrichment across LOCKs in the peritonealsubclone, whereas these same regions displayed global reductions ofH3K9Me2 in the distant metastases and their matched primary tumorsubclones (FIG. 2A, average: 591 Mb/1,470 Mb; range: 204-1,110 Mb/1,470Mb; p<2.2e-16 by chi square; Table 1 and Supplementary Data 3). Incontrast, high global levels of H3K27Me3 were detected from theseregions across all subclones (FIG. 2A), similar to the western blotfindings (FIG. 1G). We also detected patient-specific patterns of globalH3K9Me3 reprogramming in LOCKS (FIGS. 10A-10B). In patient A38, H3K9Me3was largely absent from A38Lg LOCKs relative to A38Per (loss of H3K9Me3from 184/208 Mb, 88.5%, p<2.2e-16). Loss of LOCK-wide H3K9Me3 was alsodetected between the paired primary tumor subclones in patient A13 (106Mb/118 Mb, 90.0% in A13Pr2 vs. A13Pr1, p<2.2e-16), and similar towestern blot findings (Supplementary FIG. 1C) this change was inheritedin the distant metastatic subclone (152 Mb/169 Mb, 90.2% in A13Lg vs.A13Pr1, p<2.2e-16). Finally, localized reprogramming events were alsodetected specifically within chromatin encoding differentially expressed(DE) genes in LOCKs (FIGS. 11A-11B, Supplementary Data 3). This includedreciprocal changes in H3K27Ac and H3K9Me2 over promoters coupled tosimilar reciprocal changes in H3K36Me3 and H3K27Me3 over gene bodies ofLOCK genes that were up- and down-regulated. This suggested that DEgenes from LOCKs were situated in specific sub-regions that possessedgene regulatory potential, consistent with hybrid LOCK-euchromatinislands (LOCK-EIs). Collectively, these findings indicated thatheterochromatin domains (LOCKs) represented a major target for bothglobal and local chromatin reprogramming events during the evolution ofdistant metastasis.

Because LOCKs correspond to a subset of block-like regions that are DNAhypomethylated in pancreatic and other human cancers, we also askedwhether DNA methylation changes were targeted to LOCKs during PDACsubclonal evolution. For this analysis, we performed WGBS on all of thecell lines with ChIP-seq data reported above. Because frozen tissuescorresponding to the cell lines had been exhausted during previousstudies, we selected 7 other frozen tissue samples for in vivo WGBS thatwere uniquely matched to the same formalin-fixed tissues with IHC datapresented in FIG. 1A (A124, local-regional spread) and FIG. 1C (A125,distant metastasis). Normal pancreas was included with frozen tissuesamples as an internal control. All samples and results of WGBS withcorresponding IHC, western blot, and ChIP-seq findings are summarized inTable 1, and quantified WGBS results with statistical analyses arepresented in Tables 2 and 3. These experiments revealed significantreductions in LOCK-wide DNA methylation across cell lines isolated fromdistant metastases relative to peritoneal carcinomatosis (FIGS. 2B-2C,Table 3). These findings matched the reductions of H3K9Me2 in LOCKsdetected by ChIP-seq on the same samples (FIG. 2A), revealing thatreprogramming of DNA methylation in hypomethylated block regions istargeted to LOCKs with reprogrammed histone methylation (Table 1).Analysis of the same LOCK regions from the frozen tissue samples alsorevealed relatively high DNA methylation in LOCKs from patient A124(peritoneal spread) and the founder clone from patient A125, while theprimary tumor and distant metastatic subclone descendants displayedstriking loss of DNA methylation that was even more pronounced than thatseen in the cell lines (FIGS. 2B-2C, Table 1, and Table 2). We alsodetected strong, localized DNA hypomethylation from down-regulated DEgenes in the hybrid LOCK-EI sub-regions, while up-regulated genesremained hypermethylated with sharp dips at the 5′-ends of genes,similar to H3K9Me2 (FIGS. 11A-11B). Thus, DNA methylation was globallyand locally reprogrammed across LOCKs from primary tumor and distantmetastatic subclones, similar to histone modifications. Based on thecollective immunostain (FIGS. 1A-1E), western blot (FIGS. 1F-1G),ChIP-seq (FIG. 2A), and WGBS (FIGS. 2B-2C) data (Summarized in Table 1),we conclude that a substantial fraction of global reprogramming eventswas targeted to heterochromatin domains (LOCKs) during the evolution ofdistant metastasis.

TABLE 2 Percent CpG Methylation levels across LOCK domains detected infrozen tissue samples by WGBS. DNA methylation levels were relativelyhigh in both primary tumor and metastatic tumors from patient A124, whopresented with peritoneal carcinomatosis. Similar high levels of DNAmethylation were also detected in the founder clone from patient A125,which were significantly reduced in the primary tumor subclone thatseeded distant metastases and in the liver metastases themselves.P-values were calculated with paired wilcox tests using a 3% threshold.Samples Global LOCK Methylation Sample name % CpG Methylation inSignificant Difference Sample source LOCKs by WGBS p-value vs. A124PrA124PrF 75.37% N/A (Highest Primary Tumor Methylation) A124Per 74.17% 1Peritoneal Met A125PrF 73.65% 1 Primary Tumor #1 (Founder Clone) A125PrS51.12% 2.20E−16 Primary Tumor #2 (Subclone) A125Lv1 56.95% 2.20E−16Liver Met #1 A125Lv2 59.47% 2.20E−16 Liver Met #2 Normal Pancreas 67.75%2.20E−16

TABLE 3 Percent CpG Methylation levels across LOCK domains detected incell lines by WGBS. DNA methylation levels were highest for theperitoneal subclone A38Per across cell lines. Methylation wassignificantly reduced in distant metastases from the same patient(A38Lv, A38Lg) and in primary tumor precursors (A13Pr1/2) and thematched lung metastasis from patient A13. P-values were calculated withpaired wilcox tests using a 3% threshold. Samples Global LOCKMethylation Name % CpG Methylation in Significant Difference SourceLOCKs by WGBS p-value vs. A38Per A38Per 78.58% N/A (Highest PeritonealMet Methylation) A38Lv 67.39% 2.20E−16 Liver Met A38Lg 71.67% 2.20E−16Lung Met A13Pr1 72.46% 2.18E−07 Primary Tumor 1 Subclone A13Pr2 73.48%2.14E−06 Primary Tumor 2 Subclone A13Lg 71.30% 2.20E−16 Lung Met

We next analyzed reprogramming within ECDs, which were defined byenrichments for global euchromatin modifications H3K27Ac and H3K36Me3with depletion of heterochromatin modifications (FIG. 12 ). Similar toheterochromatin, thousands of these domains (range: 1,935-2,318) werepartitioned into large, block-like segments (median lengths: 207 Kb-277Kb) that occupied similar lengths of the genome across subclones(average: 29% of the genome; range: 23.5%-32.0%, Supplementary Data 2).All subclones displayed similar global patterns of modifications withinECDs, including broad H3K36Me3 signals over gene bodies that wereflanked by sharp peaks of H3K27Ac and dips in DNA methylation at generegulatory elements, consistent with actively transcribed euchromatin(FIG. 2D). However, mapping DE genes from RNA-seq data to ECDs(Supplementary Data 3) identified clear patterns of local reprogrammingevents within chromatin encoding these genes (FIG. 2E, SupplementaryData 3). Genes up-regulated from ECDs acquired increased levels of bothH3K36Me3 and H3K27Ac, which could reflect a permissive chromatin stateor hyperactive transcription. In contrast, down-regulated genesdisplayed greatly reduced H3K36Me3 with relatively minor reductions ofH3K27Ac, which could reflect an inactive yet poised chromatin state ordirect transcriptional repression. Unlike LOCKs, DNA methylationremained stable around DE genes in ECDs (data not shown). Thus,reprogramming in ECDs was largely localized and targeted to H3K27Ac andH3K36Me3 in chromatin encoding DE genes.

Finally, we also detected patient-specific reprogramming targeted to aunique subset of very large LOCK domains. Although these regions weresituated within DNA hypomethylated blocks similar to other LOCKS, theydiffered in several other respects. First, these domains weresubstantially larger (median lengths: 730 Kb-1,340 Kb vs. 232-311 Kb forother LOCKs, Supplementary Data 2). Second, they were strongly enrichedwith H3K9Me3 yet depleted of H3K9Me2/H3K27Me3 (FIGS. 13A-13E andSupplementary Data 2). Third, their abundance was patient-specific:subclones from patient A13 possessed very few of these domains (range:50-111 domains covering 1.4-3.5% of the genome) while they occupied amuch higher fraction of the A38 genome (range: 226-344 domains covering14.5-20.6% of the genome, FIG. 13A and Supplementary Data 2). Finally,unlike reprogramming changes detected in other LOCKs (loss of H3K9Me2/3and DNA methylation), reprogramming in these LOCKs was characterized byloss of H3K9Me3 coupled to increased H3K9Me2 and DNA methylation (FIGS.13B-13E). Although the functional significance of these findings isuncertain, they could hold implications for patterns of genomeinstability that emerged during subclonal evolution, as outlined below.

Reprogrammed Chromatin Domains Specify Malignant Heterogeneity

Subclonal evolution may generate significant phenotypic heterogeneitywithin an individual patient, and we have hypothesized that suchdiversity could be encoded by large-scale epigenetic changes similar tothose detected above. We therefore wished to investigate in-depthwhether reprogrammed chromatin domains might encode heterogeneousmalignant properties between PDAC subclones from the same patient. Tothis end, we selected matched peritoneal and lung metastasis subclonesfrom the same patient (A38Per and A38Lg), performed gene ontology (GO)analyses on reprogrammed LOCK and ECD genes that were differentiallyexpressed between the subclones (Tables 4-7, derived from reprogrammedgenes as shown in FIG. 2E and FIGS. 11A-11B), and then tested whether GOresults matched actual phenotypic differences measured by experimentalassays. This analysis revealed that reprogrammed LOCKs and ECDs encodedsubstantial phenotypic differences that emerged during subclonalevolution, as described below.

TABLE 4 GO analysis of DE genes that were up-regulated from reprogrammedLOCKs in A38Lg, relative to A38Per. Genes involved in redox balance(oxidation-reduction, NADP) and EMT (cell adhesion, migration) wereup-regulated from reprogrammed DE genes in LOCKs (Detailed inSupplementary Data 3). GO Terms # of Genes % of Genes P-valueOxidation-reduction 64 6.0 3.9e−6 Oxidoreductase 56 5.2 4.7e−6 EGF-likedomain 28 2.6 6.4e−5 Transferase 105 9.8 1.0e−4 NADP 21 2.0 1.7e−4 CellAdhesion 41 3.8 1.8e−4 Cell Migration 31 2.9 2.7e−4 Cell Morphogenesis29 2.7 3.8e−4 Mitochondrion 67 6.3 4.0e−4 Acetylation 174 16.3 5.0e−4

TABLE 5 GO analysis of DE genes that were down-regulated fromreprogrammed LOCKs in A38Lg, relative to A38Per. Genes involved indifferentiation state (cell adhesion, development, epithelial genes),immune regulation (immune response, cytokines, inflammation), andresponse to environmental cues (transmembrane signaling, extracellularmatrix, secretion, locomotion) were down-regulated from reprogrammedLOCKs (Detailed in Supplementary Data 3). # of % of GO Terms Genes GenesP-value Signal 399 32.4  1.5e−31 Glycoprotein 488 40.0  1.0e−30Disulfide Bond 352 28.5  1.0e−25 Secreted 217 17.6  1.5e−18 Membrane 58247.2  1.7e−15 Polymorphism 951 77.1  1.5e−13 Immune Response 106 8.6 1.6e−13 Immunoglobin Domain 75 6.1  8.5e−11 Cytokine-Cytokine ReceptorInteraction 54 4.4  2.3e−10 Ion Channel 55 4.5 2.1e−9 InflammatoryResponse 55 4.5 7.6e−9 Cell Adhesion 93 7.5 1.4e−8 Developmental Protein98 7.9 4.4e−8 Cell Motion 69 5.6 4.7e−8 Transmembrane Protein 82 6.73.4e−7 Protease Inhibitor 25 2.0 3.9e−7 Response to Wounding 71 5.87.0e−7 Extracellular Matrix 40 3.2 1.2e−6 Epithelial CellDifferentiation 28 2.3 1.6e−6

TABLE 6 GO analysis of DE genes that were up-regulated from reprogrammedECDs in A38Lg, relative to A38Per. Genes involved in post-translationalmodifications, cell cycle control, DNA repair, response to stress, andDNA/RNA/protein biosynthesis were up-regulated from reprogrammed ECDs(Detailed in Supplementary Data 3). % of GO Terms # of Genes GenesP-value Acetylation 622 21.7 1.1e−59 Phosphoprotein 1290 45.0 5.1e−53Cell Cycle 154 5.4 3.4e−30 Mitotic Cell Cycle 137 4.8 4.9e−30 OrganelleFission 95 3.3 3.7e−25 DNA Metabolic Process 157 5.5 9.4e−25 DNA Repair95 3.3 1.4e−17 Response to DNA Damage Stimulus 113 3.9 3.8e−17 DNAReplication 68 2.4 2.7e−14 Cellular Response to Stress 144 5.0 3.1e−14Protein Biosynthesis 62 2.1 2.8e−12 ATP Binding 256 8.9 1.3e−11Ribonucleoprotein 78 2.7 4.1e−11 Nucleotide Binding 309 10.7 4.3e−11Translation 86 3.0 2.5e−9  ncRNA Metabolic Process 66 2.3 3.5e−9 Mitochondrion 167 5.8 4.3e−9  DNA Recombination 39 1.4 4.6e−9 Microtubule-based Process 70 2.4 5.9e−9 

TABLE 7 GO analysis of DE genes that were down-regulated fromreprogrammed ECDs in A38Lg, relative to A38Per. Genes involved inoncogenic signal transduction cascades (Sh3 domains, transmembraneproteins, kinases, Ras signaling), cell motion (wounding, migration,locomotion), and cell death control (apoptosis) were down-regulated fromreprogrammed ECDs (Detailed in Supplementary Data 3). % of GO Terms # ofGenes Genes P-value Alternative Splicing 1195 49.0  1.5e−16 Sh3 Domain65 2.7  6.8e−10 Phosphoprotein 1112 45.0 1.3e−9 Membrane 964 39.4 1.4e−9Response to Wounding 117 4.8 9.0e−8 Pleckstrin Homology 71 2.9 2.8e−7Cell Migration 49 2.0 3.4e−7 Small GTPase Signal Transduction 65 2.73.9e−7 Locomotion 52 2.1 1.4e−6 Intracellular Signaling Cascade 227 9.33.5e−6 Tyrosine Protein Kinase 34 1.4 5.4e−6 Regulation of Apoptosis 1536.2 1.1e−5 Protein Kinase Cascade 81 3.3 1.4e−5 Protein Amino AcidPhosphorylation 130 5.3 1.8e−5 Ankyrin Repeat 56 2.3 3.7e−5 Ras ProteinSignal Transduction 51 2.1 4.6e−5

First, a large number of DE genes involved in redox(oxidation-reduction) balance were up-regulated from reprogrammed LOCKsin A38Lg (Table 4, Supplementary Data 3). This subclone was accordinglyhighly resistant to H₂O₂-mediated oxidative stress (FIG. 3A) andpossessed higher oxidoreductase activity and NADPH levels than A38Per(FIGS. 14A-14B). Second, genes encoding differentiation state(epithelial vs. EMT) were reciprocally expressed from A38Per and A38LgLOCKs (Table 5, Supplementary Data 3), and we confirmed severalwell-known epithelial and EMT expression changes (e.g., CDH1/E-cadherin,CDH2/N-cadherin) at the protein level by western blots (FIG. 3B).Further consistent with GO results, A38Per maintainedwell-differentiated (epithelial) morphology while A38Lg was poorlydifferentiated (EMT-like) across multiple in vitro culture conditions(FIG. 14C), and immunofluorescence experiments showed that EMT emergedin the primary tumor subclone that seeded the A38Lg metastasis in vivo(FIG. 14D). We also note that immune-related genes were differentiallyexpressed from reprogrammed LOCKs (Table 5), which could holdimplications for PDAC immunotherapy. Third, genes involved in DNA repairand cell stress responses were significantly up-regulated in ECDs fromA38Lg, including genes crucial for maintenance of genome integrity(Fanconi anemia complex, non-homologous end joining, and theTOP2B/OGG1/KDM1A complex, among others, Table 6, Supplementary Data 3).This subclone was accordingly highly resistant to PDAC chemotherapy(gemcitabine) compared to A38Per (FIG. 3C), and western blots showedhyper-phosphorylation of histone H2AX S139 (γH2AX, a signature ofactivated DNA repair pathways, FIG. 3D). Fourth, genes involved inoncogenic signal transduction cascades were down-regulated in ECDs fromA38Lg, especially KRAS/ERK-related genes (Table 7, Supplementary Data3). Indeed, A38Lg showed loss of phosphorylated ERK (FIG. 3E),resistance to ERK inhibition (FIG. 3F), and minimal response toknockdown of oncogenic KRAS in 3D tumor forming assays (FIG. 3G, FIGS.14E-14F), despite possessing identical KRAS^(G12V) mutations as A38Per.Finally, mapping previously reported rearrangements from this patient tochromatin domains revealed that rearrangements were preferentiallytargeted to ECDs and the small subset of uniquely reprogrammed largeLOCK domains, whereas other LOCKs were strongly depleted (FIGS.15A-15C).

Thus, reprogrammed chromatin domains collectively specified malignantgene expression programs, divergent phenotypic properties, and patternsof genome instability that emerged during subclonal evolution in patientA38. This patient was unusual in having received chemotherapy prior totissue harvesting and had a missense mutation in SMARCA2 of unclearsignificance (CID, unpublished observations), and thus in this caseepigenetic selection may have occurred downstream of a genetic driver.Although the nature and extent of such findings will certainly varyamong patients, they imply that PDAC is capable of acquiring substantialepigenetic and malignant diversity during subclonal evolution, even inthe same cancer from the same patient.

Anabolic Glucose Metabolism Controls Epigenetic State and Tumorigenicity

We next asked whether a recurrent, metastasis-intrinsic pathway mighthave been selected for during subclonal evolution to exert upstreamcontrol over global epigenetic state and tumorigenic potential. Severalrecent studies have linked nutrient status and metabolic activity toglobal levels of histone modifications. Because distant metastases inthe rapid autopsy cohort were largely isolated from organs (liver, lung)that provide a rich supply of glucose, we asked whether reprogrammedchromatin and tumorigenicity in these subclones might have evolved adependence on specific aspects of glucose metabolism.

Altered glucose metabolism (i.e., Warburg effect) is a well-knownproperty of neoplastic and highly proliferative cells. Although most ofour metastatic subclones actually displayed modest proliferative ratesin culture (e.g., FIGS. 8A-8E) and in vivo, we nonetheless asked whetherdistant metastases might have acquired further adaptations in glucosemetabolism. Surprisingly, relative to proliferative (immortalized)normal HPDE cells and local-regional PDAC samples, glucose stronglystimulated metabolic (oxidoreductase) activity across distant metastaticsubclones (FIG. 4A), and glucose was accordingly required for thesesubclones to withstand oxidative stress (FIGS. 4B-4C). Distantmetastases also hyper-consumed glucose, as we detected elevated glucoseuptake and lactate secretion in distant metastases and their precursorsrelative to peritoneal carcinomatosis (FIG. 16A). To determine if excessglucose uptake was specifically incorporated into downstream metabolicpathways, we selected paired peritoneal and distant metastatic subclonesfrom the same patient, incubated them with ¹³C[1-2]-labeled glucose, andmeasured glucose incorporation into metabolic products with liquidchromatography followed by high resolution mass spectrometry (LC-HRMS).These experiments revealed elevated incorporation of both C1- andC1,2-labeled glucose into lactate and nucleotides in the distantmetastasis (FIGS. 4D-4E), consistent with enhanced glucose entry intoboth glycolysis and the pentose phosphate pathway (PPP).

We next asked whether distant metastases might have evolved a dependenceon specific enzymatic steps in either of these glucose-driven pathways,which we hypothesized would manifest as severe depletion of metabolitesubstrate secondary to hyper-consumption. To test this, we surveyedglycolytic and PPP metabolite profiles across a diverse panel of samplesincluding HPDE cells, peritoneal carcinomatosis, distant metastases, andprimary tumor precursor subclones. Analysis of all detected glycolyticand pentose phosphate metabolites (FIG. 16B) revealed a striking,recurrent depletion of 6-phosphogluconic acid (6PG) across distantmetastases and their precursors (FIG. 4F). 6PG is the substrate for6-phosphogluconate dehydrogenase (PGD), an enzyme involved in anabolicglucose metabolism that operates within the oxidative branch of the PPP.

Glucose may enter the PPP via the oxidative (oxPPP) or the non-oxidative(noxPPP) branch of the pathway, which are thought to be uncoupled.Although some studies in other cancers have suggested that PGD is animportant oncogene, it is KRAS-mediated noxPPP activation that drivesprimary tumor growth in mouse models of PDAC. Because KRAS and otherdriver mutations are acquired early in PDAC progression and shared byall subclones that evolve thereafter, we hypothesized that PGDdependence might have been selected for specifically during theevolution of distant metastasis to maintain reprogrammed chromatin andtumorigenicity. Glucose deprivation, RNAi against PGD, and6-aminonicotinamide (6AN, a nicotinamide antimetabolite prodrug reportedto preferentially inhibit PGD) had no effect on global chromatinmodifications in the peritoneal subclone, while all treatments reversedthe reprogrammed chromatin state of the paired lung metastasis from thesame patient (FIG. 5A). PGD loss-of-function appeared specific, as PGDknockdown did not alter expression of KRAS or other PPP components (FIG.5B).

We next asked whether PGD knockdown might affect intrinsic tumor formingcapacity across a larger panel of subclones. Despite their aggressivebehavior in patients, distant metastatic subclones were unable toeffectively form metastatic tumors in immunodeficient mice, and PGD RNAiwas not toxic to any subclones grown in routine 2-D cultures (data notshown). To bypass these limitations, we treated cells with RNAi and used3-D matrigel tumor-forming assays to measure the effects of PGDknockdown on intrinsic tumor-forming capacity. PGD RNAi had minimaleffect on the ability of HPDE cells to form spheres or local-regionalPDACs to form tumors by these assays (FIG. 5C). Remarkably, PGD RNAiuniversally interfered with the ability of distant metastatic subclonesto form tumors (FIG. 5D). These findings suggested that PGD mightrepresent a therapeutic target with selectivity for PDAC distantmetastasis. Because 6AN could represent a lead compound for futuredesign of PGD targeted therapies, we stringently tested it for activityagainst distant metastases with metabolomics, western blots, multiple 3Dtumorigenic assays, RNA-seq, and ChIP experiments.

6AN treatments slowed rates of glucose consumption and lactate secretionwith no effect on glutamine consumption or glutamate secretion indistant metastatic and precursor subclones (FIG. 17A), and 6AN reversedthe previously detected high incorporation of glucose into lactate andnucleotides (FIG. 17B). Furthermore, steady state levels of glucose andmetabolites directly upstream of the PGD reaction were dramaticallyelevated in response to 6AN with corresponding reductions in downstreammetabolites (FIG. 17C), which is consistent with strong PGD inhibitionas previously reported by others.

We next tested the effects of 6AN on epigenetic state. Strikingly, 6ANtreatments quantitatively reversed several reprogrammed chromatinmodifications across distant metastatic subclones with minimal effect onnormal cells or local-regional PDACs (FIGS. 18A-18B; summarized in FIGS.6A-6B), and this effect persisted upon removal of 6AN from the media(FIG. 18C). Because these changes mirrored aspects of LOCKreprogramming, we examined the chromatin state of LOCK DE genesregulated by 6AN, as identified by RNA-seq (Supplementary Data 3, 4).This revealed that DE genes were located within the reprogrammed hybridLOCK-EI regions that possessed strong H3K27Ac and H3K36Me3, lowH3K27Me3, and sharp 5′-depletion (dips) of H3K9Me2 (FIG. 19A,Supplementary Data 3). ChIP-seq experiments on control and 6AN-treatedA38Lg cells further showed that the quantitative increase of globalH3K9Me2 was targeted to LOCK regions that were reprogrammed in A38Lg vs.A38Per (FIG. 19B), while the reduced H3K27Ac was specifically targetedto genes repressed from LOCKs with no effect on other LOCK genes orECD-regulated genes (FIG. 19C). Levels of H3K27Me3 remained stableacross all regions in response to 6AN (FIG. 19B, FIG. 19D), similar towestern blot findings. Collectively, these experiments demonstrated that6AN selectively and quantitatively targeted several chromatin changeswithin LOCKs that emerged during the evolution of distant metastasis.

Because 6AN modulated the global epigenetic state, we hypothesized thatit might also selectively block tumorigenic potential in distantmetastatic subclones, similar to PGD knockdown experiments. Strikingly,6AN selectively and strongly blocked tumor formation in distantmetastatic and primary tumor precursor subclones but not local-regionalPDACs across multiple 3D tumorigenic experimental platforms, includingsuspension tumorsphere assays (Supplementary FIGS. 20A-20C), matrigeltumor forming assays (FIG. 6B), and injection of PDAC cells intoorganotypic stroma that recapitulates aspects of in vivo patient tumors(FIG. 6C). Thus, like PGD knockdown, chemical inhibition of PGD by 6ANselectively blocked the tumorigenic potential of distant metastaticsubclones.

We next examined our RNA-seq datasets to explore whether the abovefindings might be linked to regulation of malignant gene expressionprograms. Remarkably, over half (952/1832, 52%, Supplementary Data 4) of6AN down-regulated genes from A38Lg corresponded to genes that wereover-expressed in this subclone (compared to the peritoneal subclonefrom the same patient). In addition, a large fraction of 6ANup-regulated genes also matched DE genes that were repressed (914/2122,42%, Supplementary Data 4). Even more striking, nearly one-third(255/891, 29%, Supplementary Data 4) of recurrently over-expressed genesacross distant metastatic subclones were down-regulated by 6AN.Comparative GO analyses on these gene subsets produced overlappingresults that were strongly enriched for cancer-related functions,including mitotic cell cycle control, acetylation, chromosome stability,DNA repair, cell stress responses, and anabolic/biosynthetic activities(Tables 8-10).

TABLE 8 GO Analysis of Genes that were both Recurrently Over-Expressedin Distant Metastases and Down-Regulated by 6AN, Detected by RNA-seq(Detailed in Supplementary Data 3). Go Terms # of Genes % of GenesP-value Nucleus 112 40.0 1.4e−19 Phosphoprotein 150 53.6 1.3e−18Acetylation 80 28.6 2.7e−16 DNA Metabolic Process 36 12.9 1.2e−15 CellCycle 39 13.9 3.4e−12 M-phase 29 8.9 1.8e−11 DNA Repair 22 7.9 2.8e−10DNA Replication 18 6.4 8.5e−10 Cellular Response to Stress 27 9.65.0e−8  Ribonucleotide Biogenesis 13 4.6 6.3e−6 

TABLE 9 GO analysis of recurrently over-expressed genes detected byRNA-seq in distant metastatic subclones and primary tumor precursors,relative to peritoneal carcinomatosis (detailed in Supplementary Data3). % of GO Terms # of Genes Genes P-value Acetylation 217 20.7  8.0e−28Phosphoprotein 428 40.0  5.3e−24 Protein Biosynthesis 36 3.4  1.4e−14Nucleus 256 24.4  1.8e−13 Ribonucleoprotein 41 3.9  1.4e−12 Translation47 4.5  1.5e−12 DNA Metabolic Process 53 5.1 4.5e−9 Cell Cycle 65 6.23.4e−7 Mitochondria 64 6.1 5.4e−7 DNA Repair 31 3.0 5.2e−6 NitrogenCompound Biosynthesis 33 3.1 1.1e−5 Nucleotide Binding 102 9.7 1.4e−6M-phase 33 3.1 1.4e−5 Cellular Response to Stress 47 4.5 2.5e−5 TransitPeptide 39 3.7 3.3e−5 Nucleotide Biosynthetic Process 22 2.1 5.1e−5 ATPBinding 82 7.8 5.3e−5 DNA Replication 22 2.1 7.0e−5 WD40 Repeat 24 2.31.4e−4

TABLE 10 GO analysis of DE genes detected by RNA-seq that were down-regulated in response to 6AN, compared to DMSO control cells (A38Lgsubclone, detailed in Supplementary Data 3). % of GO Terms # of GenesGenes P-value Cell Cycle 226 12.2 2.5e−57 Acetylation 465 25.2 5.6e−57Phosphoprotein 935 50.6 2.5e−55 M-phase 135 7.3 1.2e−52 DNA MetabolicProcess 153 13.2 2.6e−40 Nucleus 582 31.5 1.4e−34 DNA Replication 82 4.41.0e−33 Chromosome Segregation 45 2.4 3.1e−24 DNA Repair 88 4.5 5.6e−24Cytoplasm 444 24.0 3.5e−23 ATP Binding 221 12.0 4.1e−22 CellularResponse to Stress 125 6.8 1.6e−19 Chromosome Organization 107 5.89.3e−17 Microtubule-based Process 70 3.8 3.6e−16 Nucleotide Binding 24413.2 4.2e−16 Cytoskeleton 113 6.1 3.2e−15 Ubl Conjugation 103 5.69.5e−12 DNA Recombination 36 1.9 1.6e−11 Macromolecular Complex Assembly115 6.2 3.0e−10

The above findings led us to hypothesize that 6AN-ablation oftumorigenicity in distant metastatic subclones might be mediated throughepigenetic control of cancer-related genes important to maintaintumorigenic capacity. To validate this, we selected two candidate genesfor in-depth experiments: N-cadherin (CDH2) and topoisomerase 213(TOP2B). CDH2 and TOP2B are both thought to be important for cancerprogression, are not known to be mutated in PDAC, can be therapeuticallytargeted, were recurrently over-expressed across distant metastatic andprimary tumor precursor subclones by RNA-seq (Supplementary Data 3), andwere selectively repressed by 6AN which we confirmed with RT-PCR (FIG.6E top panels). Furthermore, CDH2 was located within a reprogrammed LOCKtargeted by 6AN, and TOP2B was located immediately adjacent to a LOCKboundary. ChIP-qPCR assays performed on control and 6AN treated cellsshowed nearly identical enrichments for H3K9Me2 and H3K27Ac across thesegene loci in the peritoneal subclone (FIG. 6E left panels and FIG. 21A).In contrast, 6AN treatments on the matched lung metastasis from the samepatient resulted in enrichment of H3K9Me2 across both loci withconcordant reductions of H3K27Ac over the CDH2 genic region (FIG. 6Eright panels, FIG. 21A). This strongly suggested that a major downstreameffect of 6AN treatments was epigenetic repression of over-expressedcancer genes. We therefore performed RNAi experiments to test whetherknockdown of these genes might be important to selectively maintaintumorigenicity. Indeed, RNAi selectively blocked 3D tumor formation indistant metastatic and precursor subclones that over-expressed CDH2 andTOP2B, with no effect on HPDE cells or peritoneal carcinomatosis (FIG.6F). Collectively, these targeted validation studies strongly supportedconclusions inferred from the sequencing data, in that inhibition ofPGD/oxPPP by 6AN selectively targeted gene expression, epigenetic state,and downstream tumorigenic functions of over-expressed cancer genes(CDH2/TOP2B).

As detailed in FIGS. 1A-1G, a global epigenetic reprogramming occurredduring the evolution of distant metastasis. The immunohistochemical(IHC) stains against H3K9Me2/3 performed on tumor sections from 6subclones collected from two patients who presented with widespreadperitoneal carcinomatosis (a: patient A124, b: patient A141) showedsimilar strong nuclear staining across all primary tumor and peritonealsubclones. Similar stains on 6 subclones from two patients who presentedwith widespread distant metastases (c: patient A125, d: patient A132)showed progressive loss of nuclear staining that initiated in primarytumor subclones that seeded metastases (middle panel) and was furtherlost (c) or stably inherited (d) in the liver metastases. Scale bars=100μm for IHC, 20 μm for IF. IHC against the indicated modificationsperformed on tumor sections representing 4 paired subclones from apatient (patient A38) that presented with both peritoneal carcinomatosisand distant metastases shows that the peritoneal precursor subclone inthe primary tumor that seeded carcinomatosis inherited strong nuclearstaining of heterochromatin modifications as seen in the parental clonethat founded the neoplasm. In contrast, the primary tumor precursorsubclone that seeded distant metastases showed cell-to-cell variation instaining, with complete loss of staining in the paired lung metastasis.Staining for the euchromatin modification H3K36Me3 remained stableacross all subclones. f, Similar to IHC on tissues (e), western blots oncells lines collected from the peritoneal subclone (Per), livermetastasis, and lung metastasis from patient A38 also showed loss ofheterochromatin modifications in distant metastatic subclones, withcorresponding increased acetylation. Levels of H3K27Me3 and H3K36Me3 didnot differ between subclones. g, Densitometry summary of western blotfindings for the indicated histone modifications across cell lines fromdistant metastatic subclones compared to peritoneal carcinomatosis (n=8biological replicates, error bars=s.e.m., *p<0.01).

As detailed in FIGS. 2A-2E, an epigenomic reprogramming of chromatindomains during PDAC subclonal evolution was observed. Representative(left panels) and total summarized (right panels) ChIP-seq experimentsrevealed loss of H3K9Me2 from LOCKs between peritoneal (A38Per) anddistant metastatic and primary tumor precursor subclones (others).H3K27Me3 remained strong in all subclones. Bisulfite-seq data on celllines (A38, A13, top panel) and frozen tissue samples (A124, A125panels) showed that samples from local regional spread and parentalclones (A38Per, A124PrF, A124Per, A125PrF) possessed hypermethylatedLOCKs. In contrast, distant metastatic subclones (A125Lv1/2, A13Lg,A38Lv, A38Lg) and their primary tumor subclones (A125PrS, A13Pr1,A13Pr2) showed hypomethylation of DNA across the same LOCK regions.Global levels of H3K36Me3, H3K27Ac, and DNA methylation within ECDs didnot show any clear differences between subclones. In contrast, distantmetastatic subclones and primary tumor subclones displayed localreprogramming of H3K36Me3 and H3K27Ac specifically over DE genes withinECDs, compared to the same DE genic ECD regions from A38Per.

As detailed in FIGS. 3A-3G, reprogrammed chromatin domains encodedivergent malignant properties. A38Lg was remarkably resistant to H₂O₂treatments compared to A38Per. MTT signals reflect cell viabilitynormalized to untreated controls. n=4 technical replicates, *p<0.03.Western blots for proteins involved in epithelial and EMTdifferentiation were differentially expressed between A38Per and A38Lg,as predicted by GO analyses of reprogrammed DE genes from LOCKs. A38Lgwas completely resistant to gemcitabine compared to A38Per, as predictedby GO analyses of reprogrammed DE genes from ECDs. MTT signals reflectcell viability normalized to untreated controls. n=4 technicalreplicates, *p<0.01. A38Lg possessed elevated levels of yH2AX by westernblot, consistent with activation of DNA repair pathways. Western blotsshowed that A38Lg lost hyper-phosphorylated ERK and f, was resistant toERK targeted therapy, compared to A38Per. MTT signals reflect cellviability, normalized to untreated controls. n=4 technical replicates,*p<0.03. A38Lg also lost sensitivity to KRAS knockdown by Matrigel®3Dtumor forming assays, compared to A38Per. n=4 technical replicates,*p<0.01.

As detailed in FIGS. 4A-4F, hyperactive glucose metabolism and 6PGdepletion were observed in distant metastatic subclones. MTT assaysperformed on equal numbers (20K) of viable, growth-arrested cells fromthe indicated subclones showed greatly elevated signal (oxidoreductaseactivity) across distant metastatic subclones, compared to HPDE andlocal-regional PDAC samples (n=4 technical replicates for each, errorbars=s.d.m., *p<10-5). Normalized cell counts for the indicated samplesincubated with (+) or without (−) 10 mM glucose and treated with 1 mMH₂O₂ as indicated (+, −) for 24h showed that normal HPDE cells weresensitive to H₂O₂ under either glucose condition (as expected), whereaslocal-regional PDAC samples were resistant to H₂O₂ irrespective ofglucose availability (n=3 technical replicates for each, errorbars=s.d.m). In contrast, distant metastatic subclones were sensitive toH₂O₂ when glucose was not present in the media (n=3 technical replicatesfor each, error bars=s.d.m, *p<0.001). Simplified schematic of¹³C-(1,2)-labeled glucose flow through glycolysis and the PPP. Glucosethat enters the oxidative branch of the PPP has one labeled carboncleaved during conversion of 6PG to Ru5P (m+1), whereas glucose thattravels through glycolysis or the non-oxidative PPP retains both labeledcarbons (m+2). Note that cross-talk allows glucose with either labelingpattern to re-enter the other pathway and incorporate. LC-MS fornucleotides and lactate showed that these downstream metabolitesacquired greatly elevated ¹³C-1,2 labels from glucose in the lungmetastasis from patient A38 (A38Lg), compared to its paired peritonealsubclone (A38Per, n=3 biological replicates, error bars=s.d.m.,*p<0.01). Steady state LC-HRMS measurements for 6PG showed eithercomplete (ND: not detected) or near complete loss of metabolite acrossdistant metastases and their precursors compared to peritonealcarcinomatosis and HPDE cells.

As detailed in FIGS. 5A-5D, a PGD-dependence in distant metastaticsubclones was observed. Western blots against indicated histonemodifications performed on paired peritoneal (A38Per) and distantmetastatic (A38Lg) subclones from the same patient showed that globallevels of reprogrammed H3K9Me2/3 and acetylation in A38Lg were reversedby removal of glucose from the media (left panel), PGD RNAi (middlepanel), and 6AN treatments (right panel). Western blots on A38Lgindicated that PGD knockdown by RNAi did not perturb expression of otherPPP components or KRAS. PGD RNAi did not affect the ability of normalHPDE cells or local-regional PDAC samples to form tumors in 3D Matrigel®assays (representative photomicrographs shown with quantified numbers oftumors/well, n=4 technical replicates for each, error bars=s.d.m.). Incontrast, PGD RNAi significantly reduced tumor formation across alldistant metastatic subclones that were available for testing from therapid autopsy cohort (n=4 technical replicates for each, errorbars=s.d.m., *p<0.01). Scale bars: 200 μm.

As illustrated in FIGS. 6A-6F, a reversal of reprogrammed chromatin,tumorigenicity, and malignant gene expression programs by 6AN wasobserved. Densitometry summary of western blots performed on 8biological replicates, error bars=s.e.m., *p<0.01. 6AN selectivelyreversed reprogrammed H3K9Me2 and acetylation across most distantmetastatic subclones, with minimal or non-recurrent effects on H3K27Me3or H4K20Me3. Densitometry summary of western blots performed on 6biological replicates, error bars=s.e.m. 6AN had minimal effects onhistone modifications across normal (HPDE, fibroblast) or local-regionalPDAC samples. 6AN ablated tumor formation in 3D Matrigel® assays (n=4technical replicates for each, error bars=s.d.m., *p<0.01, scale bars:200 μm) and 3D tumorsphere assays across distant metastatic subclones.6AN had minimal effects on local-regional PDAC samples by either assay.6AN also blocked the ability of distant metastatic subclones to formtumors when injected into 3D organotypic stromal cultures (n=3 technicalreplicates for each, error bars=s.d.m., *p<0.05; scale bars: 200 μm).Real-time RT-PCR (top panels) showed that the distant metastaticsubclone (A38Lg) over-expressed CDH2 relative to the peritoneal subclone(A38Per) from the same patient, and that expression was repressed by 6AN(n=4 technical PCR replicates from two biological replicate experiments,error bars: s.d.m., *p=0.002). ChIP assays for H3K9Me2 and H3K27Ac withPCR primers (location indicated by numbers) spaced across the 1.4 Mbchromatin domain showed that 6AN induced spreading of H3K9Me2 across thelocus with corresponding loss of H3K27Ac in A38Lg, with no effect onA38Per (n=2 biological replicates, error bars=s.e.m.). RNAi against bothCDH2 and TOP2B selectively blocked tumor formation in distant metastaticand precursor subclones that over-expressed these genes by RNA-seq, withno effect on A38Per or HPDE cells (n=4 technical replicates for each,error bars=s.d.m., *p<0.01; scale bars: 200 μm). Specific RNAi knockdownof gene expression is shown.

As illustrated in FIGS. 7A-7C, reprogrammed chromatin across distantmetastatic subclones was detected. Western blots showed minimal orinconsistent changes for the indicated histone modifications betweenlocal-regional PDAC samples, including A38Per (Per). In contrast, apanel of distant metastatic subclones showed recurrent changes inspecific modifications, compared to A38Per. Reprogramming was alsoobserved between primary tumor subclones (Pr1, Pr2) and the lung metfrom the same patient.

As illustrated in FIG. 8A-8E, the specificity of reprogrammed histonemodifications was established. Ki67 stains showed similar cell cyclerates for peritoneal and the matched lung met grown in serum, andserum-free media (SFM) arrested growth. Serial cell counts for theindicated times confirmed equal growth rates and growth arrest in SFM.GO analysis on RNA-seq data from cells cultured in serum vs. SFM furtherconfirmed growth arrest in SFM (lung data, peritoneal gave identicalresults). Western blots showed persistence of reprogrammed chromatinmodifications in serum/proliferative (−) and SFM/growth arrested (+)cells. Treatment of the peritoneal subclone with PDAC chemotherapies(Gem: Gemcitabine, G+FU: Gemcitabine+5-Fluorouracil) did not induce lossof methylation or gain of acetylation as seen between peritoneal anddistant metastases, confirming that reprogramming was unrelated totreatment effects.

As illustrated in FIG. 9 , an enrichment of heterochromatinmodifications within LOCKs was identified. Plots of ChIP-seq readdensities normalized to inputs for histone modifications (left labels)showed that heterochromatin modifications (H3K9Me2/3, H3K27Me3) wereenriched in regions that were called LOCKs (0% to 100%, bottom panellabels) for each subclone (indicated above graphs). In contrast,euchromatin modifications (H3K36Me3, H3K27Ac) were depleted from LOCKs.

As illustrated in FIGS. 10A-10B, the reprogramming of H3K9Me3 in LOCKsduring PDAC subclonal evolution was observed. ChIP-seq data from pairedperitoneal (A38Per) and lung (A38Lg) metastatic subclones detecteddramatic reduction of H3K9Me3 in A38Lg, that overlapped with H3K9Me2(which marks LOCK domains). Similar data for patient A13, which alsoshowed loss of H3K9Me3 from LOCK regions in A13Pr2/A13Lg subclones,compared to the Al 3Pr1 primary tumor subclone.

As illustrated in FIGS. 11A-11B, the local reprogramming of DE gene lociwithin LOCKs was observed. Mapping DE genes from RNA-seq (distant metsand precursors vs. A38Per) to LOCKs revealed reciprocal changes inH3K27Me3 and H3K27Ac/H3K36Me3/DNA methylation around genes downregulatedin LOCKs. The opposite changes in H3K27Me3 and H3K27Ac/H3K36Me3 weredetected from genes upregulated in LOCKs. DNA methylation remained highin these regions. P-values for each comparison are listed inSupplementary Data 3.

As illustrated in FIG. 12 , an enrichment of euchromatin modificationswithin ECDs was observed. Plots of ChIP-seq read densities normalized toinputs for histone modifications (left labels) showed that euchromatinmodifications (H3K36Me3, H3K4Me3, H3K27Ac) were enriched in regions thatwere called ECDs (0% to 100%, bottom panel labels) for each subclone(indicated above the graphs). In contrast, heterochromatin modifications(H3K9Me2/3, H3K27Me3) were depleted from ECDs.

As illustrated in FIGS. 13A-13E, the reprogramming of large LOCKs duringPDAC evolution was observed. H3K9Me3 was enriched and DNA hypomethylatedin large LOCK domains. Striking reprogramming of H3K9Me3/2 and DNAmethylation was detected in a subset of A38 large LOCKs. H3K9Me3 wasalso enriched across large HPDE LOCKs. Examples of reprogrammed domainsbetween samples were obtained.

As illustrated in FIGS. 14A-14F, malignant heterogeneity between A38subclones was discovered. Oxidoreductase capacity was measured with MTTassays performed on equal numbers of growth-arrested cells in theabsence of serum, and MTT signals normalized to total cell numbers perwell. Consistent with GO results, A38Lg possessed higher oxidoreductaseactivity. n=4 technical replicates. NADPH/NADP levels were measured withenzyme cycling assays on equal numbers of growth arrested cells. MoreNADPH/million cells were detected in A38Lg. n=2 biological replicates.A38Per and A38Lg maintained well/poorly differentiated morphology inpatient tissues and across three separate in vitro culture conditions asindicated. IF performed on fixed tissues from the primary tumor showedloss of E-cadherin with gain of vimentin in the precursor subclone thatseeded A38Lg, consistent with EMT. RNAi knockdown of KRAS blocked 3-Dtumor formation in suspension assays more efficiently in A38Per thanA38Lg. n=4 technical reps.

As illustrated in FIG. 15A-15C, it was found that rearrangements weretargeted to Large LOCKs and ECDs. Total breakpoints were notsignificantly enriched within Large LOCKs or ECDs. Unlike typical LOCKs,Large LOCK/ECD breakpoints were significantly joined to breakpoints fromhomologous domains to form rearrangements. Examples of Large LOCKrearrangements that generated translocations and amplifications wereobserved.

As illustrated in FIGS. 16A-16B, an enhanced glucose metabolism withdepleted 6PG levels across distant metastases was observed.Extra-cellular glucose consumption and lactate secretion were elevatedin distant mets relative to per. (n=3). Schematic of glycolytic(outside) and PPP (boxed) metabolites with intra-cellular metabolitelevels plotted for each sample. Data represent LC-MS signals normalizedto protein (n=3-5).

As illustrated in FIGS. 17A-17C, it was found that 6AN targets glucosemetabolism and the PGD step of the PPP. 6AN selectively slowed rates ofextra-cellular glucose consumption and lactate secretion in metastaticsubclones with no effect on glutamine/glutamate. 6AN reducedincorporation of intracellular 013-labeled glucose into metabolitesdownstream of the PPP. 6AN greatly increased metabolite levels of PGDsubstrate (6PG) and upstream metabolites (G1,5L) with correspondingreductions in downstream products.

As illustrated in FIGS. 18A-18C, it was found that 6AN selectivelymodulated the reprogrammed chromatin state of distant metastaticsubclones. 6AN treatments generally increased global H3K9Me2 withcorresponding decreased acetylation in distant metastatic subclones.Normal cells and local-regional PDACs did not show such changes. 6ANchanges persisted after 3d treatment (+) followed by removal of 6AN fromthe media (+−) for an additional 3d.

As illustrated in FIGS. 19A-19D, it was found that 6AN targetedreprogrammed LOCK regions. Mapping 6AN repressed DE genes to A38Lg LOCKsrevealed that these were located in reprogrammed LOCK-El regions.ChIP-seq on DMSO vs. 6AN treated A38Lg detected a quantitative increasein LOCK-wide H3K9Me2 from reprogrammed regions (as aligned to A38PerLOCKs). ChIP-seq also detected 6AN-reduced H3K27Ac specifically fromgenes repressed in LOCKs with unchanged H3K27Me3.

As illustrated in FIGS. 20A-20C: 6AN selectively blocked tumor formationin distant metastatic subclones. 6AN did not interfere with the abilityof local-regional PDAC samples to form tumors in 3-D Matrigel® assays orin 3-D suspension tumorsphere assays. n=2-4. In contrast, 6AN stronglyblocked the ability of distant metastatic subclones to form tumors in3-D suspension tumorsphere assays (shown) and 3-D Matrigel® assays (FIG.5B). n=4, p<0.003. Scalebars: 200 uM.

As illustrated in FIGS. 21A-21C, the reprogramming of the TOP2B locus inresponse to 6AN was observed. RT-qPCR (top panels) showed that 6ANselectively repressed TOP2B the lung metastatic subclone. Similarly,ChIP assays showed that 6AN induced spreading of H3K9Me2 across thelocus in the lung metastasis, with no effect on the paired peritonealsubclone. Representative RT-qPCR verified RNAi knockdown of CDH2 andTOP2B with minimal effect on vimentin (normalized to ERK, which wasequally expressed across all conditions).

Discussion

The first major result of this study was widespread epigeneticreprogramming during the evolution of distant metastasis in the absenceof metastasis-specific driver mutations, i.e., those not already presentin the founder clone of the primary tumor. These involved large-scalereprogramming of histone H3K9 and DNA methylation within largeheterochromatin domains (LOCKS and hypomethylated blocks), as well asregional changes in gene regulatory modifications (H3K27Ac, H3K36Me3).Second, these changes specified heterogeneous malignant properties thatemerged during subclonal evolution. In particular, evolutionarilydivergent subclones from the same patient showed changes in geneexpression from reprogrammed regions consistent with their individualmalignant properties, including oxidoreductase capacity, differentiationstate, chemoresistance, oncogene addiction, and patterns of genomeinstability. Third, it was the PGD step of the oxPPP that controlledaspects of reprogrammed chromatin and tumorigenicity in distantmetastatic subclones, as shown by metabolomics, genetic knockdown ofPGD, chemical inhibition of PGD, and knockdown of downstream targetgenes. This strongly suggests that this anabolic glucose pathway wasselected during the evolution of distant metastasis to maintainmalignant epigenetic state and tumorigenic properties.

These findings also raise several important but complex questions, whichwe are pursuing in other studies. Perhaps the most complicated pertainsto the extent of epigenetic and malignant heterogeneity betweensubclones across patients. Just to answer this in a single patient, acombination of whole-genome mapping, RNA-seq, bioinformatics, andseveral downstream experimental approaches were required. We hypothesizethat such heterogeneity is a function of evolutionary time: patients whopresent with late-stage, widely metastatic disease may possess moreepigenetic and malignant divergence between subclones in their tumorsthan patients who present with early-stage disease. This possibilityunderscores the pressing need to detect cancers early, before suchmalignant heterogeneity arises.

Also unclear are the precise mechanisms whereby PGD/oxPPP activitycontrols global epigenetic state, which are likely to be complex. Thiscould be mediated through any of the known oxPPP-dependent changes incellular metabolism, including redox balance, fatty acid biosynthesis,and/or ribose biosynthesis, any of which can affect global epigeneticstate through control of metabolite cofactors that activate or inhibitentire classes of chromatin modifying enzymes. PGD activity itself isalso complex and subject to several modes of regulation, includingtranscriptional over-expression, post-transcriptional repression,post-translational modification, protein:protein interactions, substrateavailability, feedback inhibition, cross-talk with other pathways, andsubcellular localization including a highly conserved yetuncharacterized nuclear fraction (C. Lyssiotis, personal communication).PGD-dependence may be selected for by any of these mechanisms during theevolution of distant metastasis in different patients.

A final question is how global epigenetic changes are targeted tospecific chromatin domains that encode gene expression changes duringsubclonal evolution. We hypothesize that transcription factors andchromatin modifying enzymes that directly bind these regions play majorroles in targeting the reprogramming events, and several candidates wererecurrently over-expressed in our RNA-seq datasets. This includes thehistone demethylase KDM1A (LSD1), which could be particularly importantsince we previously showed that this enzyme controls LOCK reprogrammingand other studies have shown that it regulates breast cancer metastasis.

In summary, our findings in conjunction with deep sequencing studies onmany of the same samples reported here suggest a model whereby drivermutations arise early to initiate PDAC tumorigenesis, followed by aperiod of subclonal evolution that generates heterogeneous metabolic,epigenetic, and malignant properties. Like driver mutations, thoseproperties that confer increased fitness to cells that acquire them maybe selected for and clonally expanded during invasive tumor growth andmetastatic spread. The strong oxPPP-PGD dependence we observe in distantmetastatic subclones could reflect such selection: distant metastaticsites provide ample glucose to fuel the pathway, pathway products(glucose-dependent NADPH) reduce oxygen species encountered within thesites, and the pathway itself is coupled to epigenetic programs thatpromote tumorigenesis. As such, reversal of malignant epigeneticprograms by targeting the oxPPP could represent an effective therapeuticstrategy for metastatic PDAC, one of the most lethal of all humanmalignancies.

Data Deposits

All ChIP-seq, RNA-seq, and bisulfite-seq sequencing data has beendeposited online (GEO Number: GSE63126) at the following URL:ncbi.nlm.nih.gov/geo/query/acc.cgi?token=sxyjkaqsvfalheh&acc=GSE63126

Methods Summary

Tissue samples and cell lines were previously collected from PDACpatients by rapid autopsy, sequenced-validated, and monitored formycoplasma as previously described. Low passage (2-17) rapid autopsycell lines were cultured at 37° C. in DMEM with 10% fetal bovine serum(FBS, Gibco). For MTT assays, 15,000 cells/well were plated into 96 wellplates in triplicate, treated 12 hours later, and assayed after 24 hr(glucose responses) or 6 days (chemotherapy) with CellTiter96 (Promega).For glucose response assays, nutrient-deplete DMEM (no glucose,glutamine, pyruvate, or serum) was used with addition of glucose asindicated. For glucose-dependent oxidative stress analysis, cells platedin triplicate and grown to 80% confluence followed by incubation innutrient-deplete DMEM containing 10% dialyzed FBS with or without 10 mMglucose and 1 mM H₂O₂ for 24 hours. Cells were then washed with PBS,trypsinized, and viable cells counted with a hemocytometer. Glucoseuptake and lactate secretion were measured with a YSI 7100 Bioanalyzeras described in the supplementary methods. For ¹³C-1,2 glucose tracingand steady state metabolite profiling, the Q Exactive MS (QE-MS; ThermoScientific) coupled to liquid chromatography (LC Ultimate 3000 UHPLC)was used for metabolite separation and detection as previouslydescribed. Detailed conditions are provided in the supplementarymethods.

Histones were acid extracted as described and western blots performed on3.5 ug histones, which were checked by Ponceau stains prior to westernblot to ensure equal loading. Densitometry was performed with ImageJsoftware. RNA was extracted with Trizol reagent (Life Technologies) andisopropanol precipitated. Genomic DNA was purified with MasterPure DNAextraction reagents (Epicenter). Immunohistochemistry, H&E staining, andimmunofluorescence on formalin-fixed, paraffin-embedded (FFPE) tissuemicroarray sections (TMAs) were performed according to standardprocedures. Antibodies used for western blot, IHC, and ChIP are listedin Table 11.

TABLE 11 Antibodies and conditions used for western blots, immunostain,and ChIP experiments Antibody Source Catalogue # Western dilution Immunodilution CDH1 Cell Signaling 4065 1/500 1/100, IF Vimentin NeoMarkersMs-129-P 1/500 1/100, IF G6PD Cell Signaling 8866S 1/500 N/A PGD CellSignaling 13389S 1/500 N/A TKT Cell Signaling 8616S 1/500 N/A TALDO1Santa Cruz Sc-134795 2 ug/ml N/A ERK1/2 Cell Signaling 4696S 1/500 N/Ap-ERK1/2 Cell Signaling 4370S 1/500 N/A KRAS Santa Cruz Sc-30 2 ug/mlN/A RPE Santa Cruz Sc-162124 2 ug/ml N/A RPIA Abcam Ab181235 1/500 N/ACD44 Cell Signaling 5640S 1/500 N/A Epcam Millipore CBL251 1/500 N/ACDH2 Cell Signaling 4061S 1/250 N/A H3K9Me2 Abcam ab1220 1/10,000 1/5000IHC, IF H3K9Me3 Abcam ab8898 1/10,000 1/5000 IHC H3K27Me3 Millipore07-449 1/7500 N/A H3K9Ac Millipore 07-352 1/10,000 N/A H3K36Me3 Abcamab9050 1/10,000 1/5000 IHC H3K27Ac Abcam ab4729 1/5000 N/A H4K20Me3Millipore 07-463 1/1000 1/5000 IHC H4K16Ac Millipore, 07-329, ab1094631/10,000 N/A Abcam total H3 Abcam ab 1791 1/30,000 N/A total H4 Abcam ab10158 1/30,000 N/A YH2AX Abcam ab11174 1/5,000 N/A

RNAi experiments were performed with siRNA transfections(Oligofectamine, Life Technologies) using negative control siRNA (Sigma,SIC001) and pre-designed siRNA oligonucleotides against indicated genesin parallel (Sigma, PGD: SASI_Hs02_00334150, CDH2: SASI_Hs01_00153995,TOP2B: SASI_Hs02_00311874). siRNAs against mutant KRAS^(G12V)(CUACGCCAACAGCUCCAAC) (SEQ ID NO:1) were custom designed. Cells wereincubated with siRNAs for 4 days after transfection and harvested. Fordrug treatments in 2-D, cells were grown to 70-80% confluency andtreated for 3 days with 250 uM 6AN or DMSO negative control.

3-D matrigel assays were adapted from Cheung et al. (Control of alveolardifferentiation by the lineage transcription factors GATA6 and HOPXinhibits lung adenocarcinoma metastasis. Cancer Cell 23, 725-38 (2013)).Briefly, 2-D cultures were trypsinized into single cells, 4,000 cells/mLwere suspended and thoroughly mixed in ice-cold DMEM containing 5%matrigel (BD systems) and 2% FBS (+/−DMSO/6AN as needed), 500 ul platedin quadruplicate into 24 well ultra-low attachment plates and incubatedfor at least 7 days to allow tumor growth. Well-formed tumors were thencounted and representative photographs taken with an EVOS instrument.3-D suspension tumorsphere assays were performed with 20,000 startingcells/well in ultra-low attachment 6 well plates as described, andtumors counted/photographed after at least 7 days of tumor growth.

Organotypic tumor forming assays were adapted from Ridky et al. (P. A.Invasive three-dimensional organotypic neoplasia from multiple normalhuman epithelia. Nat Med 16, 1450-5 (2010)) and Andl et al. (Epidermalgrowth factor receptor mediates increased cell proliferation, migration,and aggregation in esophageal keratinocytes in vitro and in vivo. J BiolChem 278, 1824-30 (2003)). Briefly, 6 well permeable transwell plates(Costar 3414) were overlayed with 1 mL type 1 collagen containing10×DMEM (acellular layer). Human dermal fibroblasts (ATCC) weresuspended (12×10⁶ cells/mL) in a mixture of ice cold 10×DMEM, 10% FBS,52.5% collagen, and 17.5% matrigel (cellular layer), thoroughly mixed,and 2 mL/well plated over the acellular layer. The mixture was allowedto partially solidify for approximately 15 minutes at 37° C., followedby triplicate injection of 1×10⁶ PDAC (suspended in 20 ul DMEM) cellsinto the cellular layer. Cells were incubated for 24 hours in fibroblastgrowth media above and below the inserts to initiate contraction of thediscs. Fresh media with DMSO or 6AN was then added and replenished every2 days for 6 additional days, followed by addition of DMEM with DMSO or6AN underneath the inserts (no media on the top) for an additional 7days. Discs were harvested, fixed overnight in 10% formalin, thinlysectioned, paraffin embedded, and stained with H&E. Tumors werephotographed and measured with an Olympus BX53 microscope using cellSensStandard software.

Tests for statistical significance (two-tailed students t-test) wereperformed on data collected from technical replicate (performed inparallel at the same time) or biological replicate (performed atdifferent times) experiments as indicated in the figure legends usingexcel software for western blot densitometry, MTT assays, and tumormeasurements. Whole genome bisulfate sequencing and RNA-seq wereperformed with HiSeq instruments (Illumina) as described in Hansen etal. (Increased methylation variation in epigenetic domains across cancertypes. Nat Genet 43, 768-75 (2011)). ChIP assays were performed aspreviously described for fixed cells with sonication. For ChIP-qPCR,equal amounts of paired input/IP DNA were amplified by real-time PCR(Roche LightCycler96) and fold enrichments calculated. Primer sequencesare listed in Supplementary Table 11. For ChIP-seq, immunoprecipitatedand input DNA was further sheared to 200-300 bp fragments, size-selectedon agarose gels, and sequenced on either HiSeq (Illumina) or SOLiD(Applied Biosystems) formats with comparable results. IP sequencingreads were normalized to their corresponding inputs. Sequencingprocedures, bioinformatics methods including domain calls, andstatistical analyses are described in detail within the supplementarymethods section.

TABLE 12 Real-time PCR primer sequences used forChIP-qPCR and RT-PCR experiments Locus/AssayPrimer Name: Sequence (5′-3′) SEQ ID NO: CDH2 ChIPChr18_24.258F: GCTCAGCCCTGTATCAGCCAGC  2 CDH2 ChIPChr18_24.258R: GGGTTACAGGTATGAGCCACTGC  3 CDH2 ChIPChr18_24.506F: AATGGAGAAGTCAGGAATGTAGTCC  4 CDH2 ChIPChr18_24.506R: GTATTTCATTTATCAAGTTGCAGCTCC  5 CDH2 ChIPChr18_24.834F: TTTGCTTCTCACTCCAAGTTCATCC  6 CDH2 ChIPChr18_24.834R: CAACCTCAGGAACAATGCATCAGC  7 CDH2 ChIPChr18_25.125.6F: CGAAACAGTCCAGCTGCTATGG  8 CDH2 ChIPChr18_25.125.6R: CTTGGCTATTGTGACTGGTACTGC  9 CDH2 ChIPChr18_25.428.000F: CCAATGCACTAATTTAATGTCATGC 10 CDH2 ChIPChr18_25.428.000R: CGTGCTAATTTCTATGGTACACTGG 11 CDH2 ChIPChr18_25.632F: CCTAATCCAATATGCCTGGTGTCC 12 CDH2 ChIPChr18_25.632R: CTGGAAGTCTGAGATCAAGGTGC 13 CDH2 ChIPChr18_25.778F: AATAATCACGAAGCACTTCTGTATTGC 14 CDH2 ChIPChr18_25.778R: TCACCAGCAGACATAGTCATACTTCC 15 CDH2 ChIPChr18_25.808F: CCTTGGAGGTGGAGTCTACAGAGG 16 CDH2 ChIPChr18_25.808R: CTGCTAGCGTAGCCATCTGAGATCG 17 TOP2B ChIPChr3_25.398F: GCCCTGTCTTCCCAGAATCATTGC 18 TOP2B ChIPChr3_25.398R: CATGAAGCCTATGAAGATCATTATGG 19 TOP2B ChIPChr3_25.540F: TTTAGCCAGCAAGTATTCTAGCATGG 20 TOP2B ChIPChr3_25.540R: GTCAGTGTGATTCAGTAACAATGATGG 21 TOP2B ChIPChr3_25.622F: CCTGCTCAAGGCTGACATGTCACC 22 TOP2B ChIPChr3_25.622R: GTCGGACTCGATGGTCAGCACTGG 23 TOP2B ChIPChr3_25.733F: AACCCGAAACTTTCAATGCACTTGG 24 TOP2B ChIPChr3_25.733R: CTTCCTCTATAGTGAAGACCCTAGG 25 TOP2B ChIPChr3_25.812F: TATGGCCATTCTTGCAGCAGTAAGG 26 TOP2B ChIPChr3_25.812R: AAAGTTGGCTAAGGACATGAATAGGC 27 TOP2B ChIPChr3_25.973F: GGAGATTCCCTCAGGTGCCTATACC 28 TOP2B ChIPChr3_25.973R: CTGGTGTTCCAGGCACCACTGAGG 29 CDH2 RT-PCRCDH2F: TTATTACTCCTGGTGCGAGT 30 CDH2 RT-PCR CDH2R: GAGCTGATGACAAATAGCGG31 TOP2B RT- TOP2BF: GTTACAGGTGGTCGTAATGGTT 32 TOP2B RT-TOP2BR: TTGGCTTCAGAAGTCTTCATCA 33

Supplementary Methods

YSI metabolite analysis. Metabolite consumption (glucose and glutamine)and production (lactate and glutamate) were measured using a YSI 7100Bioanalyzer. Indicated cell lines were plated at day −1 in a 6-wellplate. At day 0 cells were counted (3 wells) or cultured in eitherregular medium or medium supplemented with the indicated compound.Tissue culture supernatants (1 mL, n=3, each condition) were harvested72 hours after cell plating. Tissue culture conditions were optimized toensure nutrient availability and exponential cell growth. Metaboliteconsumption/production data were normalized to cell number area underthe curve, as previously described (Lee et al 2014: PMID: 24998913). Thearea under the curve (AUC) was calculated as N(T)d/ln2(1-2^(−T/d)),where N(T) is the final cell count, d is doubling time, and T is time ofexperiment. Doubling time was calculated as d=(T)[log(2)/log(Q2/Q1)],where Q1 is starting cell number and Q2 is final cell number, asdetermined by manual counting using a hemocytometer.

LC-HRMS Metabolite Profiling. LC-HRMS samples were prepared and analyzedas described in Liu et al. (Development and quantitative evaluation of ahigh-resolution metabolomics technology. Anal Chem 86, 2175-84 (2014)).For glucose tracing experiments, cells were plated into 6 well plates intriplicate, grown in DMEM with 10% FBS until 70-80% confluent, washed 2×with nutrient deplete DMEM, and incubated in nutrient deplete DMEMcontaining 10 mM ¹³C-1,2 labeled glucose (Cayman) and 10% dialyzed FBS(Invitrogen) for an additional 36 hours. Additional replicates were alsoincluded and counted at the end of the experiment for normalization.Metabolism was quenched by quickly removing media and adding 1 mLpre-chilled (−80° C.) LC-MS grade 80% methanol (Sigma), incubated at−80° C. for at least 20 minutes, followed by scraping into the methanoland pelleting of metabolites by centrifugation. For drug treatments,cells were incubated in standard DMEM+/−DMSO/6AN for 36 hours, followedby incubation in labeled glucose media+/−DMSO/6AN for an additional 36hours, quenched and pelleted as above. Pellets were reconstituted inequal volumes of 1:1 LC-MS grade acetonitrile:methanol and water and 5ul were injected to the LC-QE-MS for analysis. For steady statemeasurements cells were incubated in growth media (DMEM with 10% FBS forPDACs, keratinocyte serum-free media for HPDE) until they reached 80-90%confluence, followed by 48 hours in DMEM without serum (for PDACs, sincethe standard growth media for comparison HPDE cells also did not containserum). Metabolism was then quenched with methanol and metabolitespelleted as above. Pellets were reconstituted into a volume normalizedto protein content (15 uL of 1:1 acetonitrile:methanol and 15 uL ofwater was used per 1 mg protein) and analyzed by LC-QE-MS. Raw datacollected from the LC-QE-MS was processed on Sieve 2.0 (ThermoScientific) using a targeted frame-seed that included glycolytic/PPPmetabolites as required for the analysis. The output file includingdetected m/z and relative intensity in different samples is obtainedafter data processing, and replicates of selected metabolites from eachsample were graphed and presented as shown in the figures.

Preparation of sequencing libraries. Libraries were prepared from 2-10ng of IP ChIP DNA and 100 ng of input DNA and sequenced on IlluminaHiSeq (APF laboratory). Briefly, samples were checked for quality andconcentration from 150-250 bp on a bioanalyzer. DNA was end-repairedusing Klenow polymerase in 58 ul of reaction buffer. For IP DNA, Klenowwas diluted 1:5. Samples were incubated at 20° C. for 30 minutes andsubsequently purified on QIAquick PCR purification columns. A-tails werethen added to the DNA with Klenow and dATP in NEB buffer 2 at 37° C. for30 minutes and cleaned with Qiagen MiniElute PCR purification columns.Sequencing adapters were then ligated onto the DNA for 15 minutes atroom temperature followed by cleaning with MiniElute columns. Sampleswere then run on 2% agarose gels and DNA from 216 bp-366 bp (DNA plusadapters) were cut from the gel and purified with Qiagen Gel extractionkits. Concentrations were then checked on a bioanalyzer and 8 ng werePCR amplified with Phusion polymerase (Fisher) for 15 cycles (10 sec 98°C., 30 sec 65° C., 30 sec 72° C.) followed by 5 minutes at 72° C.Samples were then cleaned with Ampure kits (Illumina) and washed with80% ethanol. DNA samples were resuspended at the end of the cleanup into17.5 ul buffer EB (Qiagen) and subjected to next generation sequencingon Illumina HiSeq platform according to manufacturer's instructions. ForSOLID sequencing, ChIP DNA was prepared and samples were processedaccording to manufacturer's protocols in the Johns Hopkins CRBII corefacility.

BS-Seq data processing. 100 bp paired-end HiSeq2000 sequencing readswere aligned by BSmooth bisulfite alignment pipeline (version 0.7.1) aspreviously described in Hansen et al. (Increased methylation variationin epigenetic domains across cancer types. Nat Genet 43, 768-75 (2011)).Briefly, reads were aligned by Bowtie2 (version 2.0.1) against humangenome (hg19) as well as the lambda phage genome. After alignment,methylation measurements for each CpG were extracted from aligned reads.We filtered out measurements with mapping quality <20 or nucleotide basequality on cytosine position <10 and we also removed measurements fromthe 5′ most 10 nucleotides of both mates. Then, bsseq package in BSmoothwas used to identify small and large differentially methylated regions(DMRs). Only CpGs with at least coverage of 3 in all samples wereincluded in our analysis. For small DMRs, smooth window of 20 CpGs or 1kb was used, and t-statistic cutoff of −4.6, 4.6 and methylationdifference greater than 20% were used for identifying small DMRs. Whilefor large DMRs, smooth window of 200 CpGs or 10,000 bps was used, andt-statistic cutoff of −2, 2, methylation difference greater than 10% andlength of DMRs >5 kb were used for identifying large DMRs.

RNA-Seq data processing. 100 bp paired-end HiSeq2000 sequencing readswere aligned against human genome (hg19) by OSA (version 2.0.1) withdefault parameters. After alignment, only uniquely aligned reads werekept for further analysis. Gene annotation information was downloadedfrom ENSEMBL (ensembl.com, release 66). Reads count for each gene of allsamples were estimated using HTSeq(huber.embl.de/users/anders/HTSeq/doc/overview.html) and then were usedto identify differentially expressed (DE) genes using DESeq package.Genes with FDR <0.01 and fold-change >1.5 were considered DE genes.

Chip-seq data processing. For 46 bp paired-end Illumina HiSeq2000sequencing data, reads were aligned against human genome (hg19) usingBWA with default parameters as described in Li et al. (Fast and accurateshort read alignment with Burrows-Wheeler transform. Bioinformatics 25,1754-60 (2009)). After alignment, duplicate reads were removed and onlyuniquely aligned reads were kept for further analysis. For 48 bpsingle-end Solid sequencing data, reads were aligned using Bowtie⁷ withdefault parameters and only uniquely aligned reads were kept for furtheranalysis. For narrow histone modification peaks (H3K4Me3 and H3K27Ac),MACS2 were used for peak calling with default parameters⁸. For broadhistone modification enrichments (H3K36Me3, H3K27me3, and H3K9Me2/3),peak calling was performed using RSEG which is based on hidden Markovmodel (HMM) and specifically designed for identifying broad histonepeaks (see Song et al. (Identifying dispersed epigenomic domains fromChIP-Seq data. Bioinformatics 27, 870-1 (2011))).

Identifying large chromatin domains. We define LOCK domains forheterochromatin modifications (H3K9Me2/H3K27Me3) based on the peakcalling results from RSEG. Briefly, peaks shorter than 5 kb were firstremoved to prevent regions with many nearby, short peaks being called asLOCKs. Then, neighboring peaks with distance less than 20 kb were mergedto into one domain. Merged regions greater than 100 Kb identified inboth biological replicates were called LOCKs. We noticed another uniquesubset of LOCKs that were invariably larger than 500 kb, stronglyenriched with H3K9Me3, depleted of H3K9Me2 and H3K27Me3, and flanked bystrong peaks of H3K27Me3 at the boundaries. Because of this, we definedthese LOCKS by H3K9Me3 regions with length greater than 500 kb and lessthan 50% of their length overlapped with H3K27me3. Finally, the largeregions (>50 kb) between heterochromatin domains that contained at leastone gene with corresponding euchromatic H3K4Me3/H3K27Ac regulatory peakswere defined as ECDs. Because we found that H3K27Ac alone was sufficientfor these calls, H3K4Me3 was also used for the initial test dataset withA38Per/Lg, but not required in subsequent datasets (A13Pr1/2, A13Lg).All codes used for domain calls are available upon request.

Defining different gene groups. Genes were classified as belongingwithin euchromatin (>50% of genic region located in ECDs) orheterochromatin (>50% of genic region located in those heterochromatindomains including LOCKS and G-LOCKs). A handful of other genes that didnot fit these criteria and were classified as “other”.

Quantifying and enrichment plotting of ChIP-seq and RNA-seq. To ploteach histone modification on defined large chromatin domains and theirflanking regions, we divided flanking sequences of chromatin domainsinto bins with fixed length (in bp) and domains themselves into binswith fixed percentage of each domain length. ChIP enrichment wasmeasured and normalized as described in Hawkins et al. (Distinctepigenomic landscapes of pluripotent and lineage-committed human cells.Cell Stem Cell 6, 479-91 (2010)). In brief, the number of reads perkilobase of bin per million reads sequenced was calculated for each ChIPand its input control (denoted as RPKM_(chip) and RPKM_(input)). ChIPenrichment is measured as ΔRPKM=RPKM_(ChIP)−RPKM_(input) and ChIPenrichment regions should have ΔRPKM>0. Then all ΔRPKM were normalizedto a scale between 0 and 1 and the average normalized ChIP enrichmentsignals across all large chromatin domains were plotted for each histonemark. RNA-Seq data was also normalized by the number of reads perkilobase of bin per million reads sequenced and plotted similarly.

Supplementary Data

Supplementary Data 1. This lists numbers of sequencing reads (totalreads and uniquely aligned reads) for all replicate samples forChIP-seq, WGBS, and RNA-seq experiments and includes correlationcoefficients between the replicate samples.

Supplementary Data 1A: Summary of ChIP-seq reads for all replicatesamples

Samples (Name_replicate#_IP Uniquely antibody) Total reads aligned reads38Per_1_K27ac 27,100,820 23,396,028 38Per_2_K27ac 20,750,076 17,913,27838Lg_1_K27ac 22,821,996 19,946,514 38Lg_2_K27ac 25,996,970 22,475,05838Per_1_K9ac 27,148,594 22,200,344 38Per_2_K9ac 25,585,898 18,706,45638Lg_1_K9ac 26,607,612 21,750,232 38Lg_2_K9ac 28,316,524 23,222,23038Per_1_K4me3 28,059,734 23,718,542 38Per_1_K4me3 43,068,697 20,704,38338Lg_2_K4me3 24,455,456 21,184,788 38Lg_2_K4me3 44,715,633 17,458,60338Per_1_K36me3 44,738,783 23,961,134 38Per_2_K36me3 25,140,43419,239,814 38Lg_1_K36me3 25,532,398 18,263,078 38Lg_2_K36me3 48,929,33624,747,992 38Per_1_K27me3 26,546,612 21,523,318 38Per_1_K27me347,076,156 21,613,912 38Lg_2_K27me3 25,528,442 20,886,386 38Lg_2_K27me344,444,667 16,568,247 38Per_1_K9me2 69,629,048 55,830,176 38Per_K9me2_264,949,506 51,281,848 38Lg_K9me2_1 51,787,694 39,725,736 38Lg_2_K9me271,421,136 55,643,850 38Per_1_K9me3 28,530,078 18,843,038 38Per_2_K9me330,869,994 19,860,544 38Lg_1_K9me3 33,055,178 20,251,534 38Lg_2_K9me336,205,418 22,568,940 38Per_1_Input 28,748,102 22,637,100 for 38Per_1K16ac, K27ac, K9ac, K4me3, K27me3, K9me2 and K9me3 38Per_1_Input43,698,893 20,955,594 for 38Per_1 Solid K4me3, K36me3 and K27me338Per_1_Input 78,376,368 61,062,024 for 38Per_1 K9me2 38Per_2_Input26,162,800 20,358,392 for 38Per_2 K16ac, K9ac, K36me3, K9me2 and K9me338Per_2_Input 22,795,752 17,717,432 for 38Per_2 K27ac 38Per_2_Input73,425,982 57,237,686 for 38Per_2 K9me2 38Lg_1_Input 30,391,44423,128,054 for 38Lg_1 K16ac, K27ac, K9ac and K9me3 38Lg_1_Input26,527,468 20,878,592 for 38Lg_1 K36me3 and K9me2 38Lg_1_Input56,918,912 44,006,646 for 38Lg_1 K9me2 38Lg_2_Input 24,372,67021,204,480 for 38Lg_2 K16ac, K27ac, K9ac, K4me3, K27me3 and K9me338Lg_2_Input 43,698,893 19,884,219 for 38Lg_2 Solid K4me3 and K27me338Lg_2_Input 42,655,553 25,391,128 for 38Lg_2 Solid K36me3 38Lg_2_Input82,576,576 65,151,312 for 38Lg_2 K9me2 13Pr2_1_K27Me3 45,562,12235,405,382 13Pr2_2_K27Me3 48,647,198 37,601,820 13Pr2_1_K36Me350,525,636 40,320,884 13Pr2_2_K36Me3 43,172,826 34,777,556 13Pr2_1_K9Me352,034,354 28,398,244 13Pr2_2_K9Me3 47,846,284 27,025,984 13Pr2_1_K27Ac39,057,664 32,754,504 13Pr2_2_K27Ac 45,988,666 38,419,042 13Pr2_1_K9Me236,367,760 27,747,040 13Pr2_2_K9Me2 49,421,058 36,685,524 13Pr2_1_Input42,932,496 34,453,136 for 13Pr2_1 K27Ac and K9Me2 13Pr2_2_input39,166,036 31,527,030 for 13Pr2_2 K27Ac and K9me2 13Pr2_1_Input43,812,050 33,019,642 for 13Pr2_1 K36Me3 13Pr2_2_Input 42,483,79032,000,834 for 13Pr2_2 K36Me3 13Pr2_1_Input 36,646,218 27,841,600 for13Pr2_1 K27Me3 and K9Me3 13Pr2_2_Input 37,605,184 28,502,806 for 13Pr2_2K27Me3 and K9Me3 13Pr1_1_K27Me3 42,464,788 32,291,936 13Pr1_2_K27Me342,717,990 32,886,274 13Pr1_1_K36Me3 45,628,144 34,403,25613Pr1_2_K36Me3 47,010,260 35,169,858 13Pr1_1_K27Ac 42,969,352 36,362,08413Pr1_2_K27Ac 52,912,056 43,709,782 13Pr1_1_K9Me3 43,261,454 27,946,13613Pr1_2_K9Me3 48,134,638 30,111,834 13Prl_1_K9Me2 51,237,440 37,126,41013Pr1_2_K9Me2 44,260,552 34,079,972 13Pr1_1_K4Me3 47,817,718 40,460,33213Pr1_2_K4Me3 39,916,242 34,219,430 13Pr1_1_Input 27,015,968 21,991,728for 13Pr1_1 K9Me3 and K9Me2 13Pr1_2_Input 42,754,918 34,651,668 for13Pr1_2 K9Me3 and K9Me2 13Pr1_1_Input 37,703,540 30,589,064 for 13Pr1_1K36Me3 and K27Ac 13Pr1_2_Input 37,251,906 30,188,840 for 13Pr1_2 K36Me3and K27Ac 13Pr1_1_Input 45,330,014 36,495,018 for 13Pr1_1 K27Me3 andK4Me3 13Pr1_2_Input 41,671,660 33,507,094 for 13Pr1_2 K27Me3 and K4Me313Lg_1_K27Me3 46,174,838 35,837,518 13Lg_2_K27Me3 47,763,132 36,963,90013Lg_1_K36Me3 48,735,296 39,047,890 13Lg_2_K36Me3 44,439,570 35,973,06013Lg_1_K27Ac 52,682,716 44,187,348 13Lg_2_K27Ac 38,043,964 32,298,34413Lg_1_K9Me3 44,449,474 25,717,050 13Lg_2_K9Me3 49,020,848 28,456,69213Lg_1_K9Me2 40,580,872 31,979,380 13Lg_2_K9Me2 42,760,754 33,395,09413Lg_1_input 53,086,242 42,631,966 for 13Lg_1 K27Ac and K9Me213Lg_2_input 41,676,088 33,503,572 for 13Lg_2 K27Ac and K9Me213Lg_1_Input 40,822,392 31,354,916 for 13Lg_1 K36Me3 13Lg_2_Input45,292,342 35,189,166 for 13Lg_2 K36Me3 13Lg_1_Input 49,243,12637,741,490 for 13Lg_1 K27Me3 and K9Me3 13Lg_2_Input 47,226,32236,317,110 for 13Lg_2 K27Me3 and K9Me3 HPDE_1_K27Me3 41,619,7301,644,221,182 HPDE_2_K27Me3 45,844,562 1,870,133,582 HPDE_1_K36Me338,327,244 28,084,826 HPDE_2_K36Me3 35,519,752 27,012,400 HPDE_1_K27Ac52,294,724 43,428,888 HPDE_2_K27Ac 35,271,886 30,001,598 HPDE_1_K9Me349,006,354 31,150,780 HPDE_2_K9Me3 49,995,186 31,313,578 HPDE_1_K9Me247,415,884 36,297,824 HPDE_2_K9Me2 47,621,364 36,772,334 HPDE_1_K4Me345,262,500 39,528,934 HPDE_2_K4Me3 34,511,978 30,181,046 HPDE_1_Input50,286,666 40,743,792 for HPDE_1 K9Me3 and K9Me2 HPDE_2_Input 45,780,73637,154,676 for HPDE_2 K9Me3 and K9Me2 HPDE_1_Input 36,424,754 29,600,008for HPDE_1 K36Me3 and K27Ac HPDE_2_Input 48,330,362 39,054,926 forHPDE_2 K36Me3 and K27Ac HPDE_1_Input 38,269,792 30,829,930 for HPDE_1K27Me3 and K4Me3 HPDE_2_Input 37,635,368 30,421,766 for HPDE_2 K27Me3and K4Me3 38Lg_DMSO_1_K27Me3 45,364,340 33,889,440 38Lg_DMSO_2_K27Me337,628,254 29,077,804 38-5_DMSO_1_K9Me2 57,024,354 42,891,92438Lg_DMSO_1_K27Ac 44,665,878 37,664,038 38-5_DMSO_1_Input_batch469,705,942 56,571,128 for 38-5_DMSO_1 K9Me2 38Lg_DMSO_1_Input 37,296,75230,470,440 for 38Lg_DMSO_1 K27Ac 38Lg_DMSO_1_Input 49,727,864 40,284,668for 38Lg_DMSO_1 K27Me3 38Lg_DMSO_2_Input 40,505,458 32,886,902 for38Lg_DMSO_2 K27Me3 38Lg_6AN_1_K27Me3 50,310,568 39,528,06438Lg_6AN_2_K27Me3 38,884,546 32,325,978 38-5_6AN_1_K9Me2 33,324,39624,956,330 38Lg_6AN_1_K27Ac 40,895,998 34,565,99838-5_6AN_1_Input_batch4 42,480,878 34,537,520 for 38-5_6AN_1 K9Me238Lg_6AN_1_Input_batch3 40,615,920 33,033,908 for 38Lg_6AN_1 K27Ac38Lg_6AN_1_Input_batch5 45,637,332 37,039,730 for 38Lg_6AN_1 K27Me338Lg_6AN_2_Input_batch5 36,771,474 29,835,218 for 38Lg_6AN_2 K27Me3Total 7,441,856,062

Supplementary Data 1B: Summary of WGBS reads for all replicate samples

Aligned Coverage Samples Total reads Aligned reads Total CpGs CpGs perreplicate 38Per rep1 281,674,084 234,515,645 28,217,448 24,736,894 5.7438Per rep2 279,677,926 233,819,498 28,217,448 24,668,800 5.65 38Lg rep1246,354,388 242,041,867 28,217,448 24,079,382 4.96 38Lg rep2 293,066,340203,060,037 28,217,448 24,568,234 5.87 38-Lv rep1 481,671,728394,675,778 28,217,448 25,699,745 12.06 38-Lv rep2 550,721,820443,244,002 28,217,448 25,797,654 13.57 13Pr2 rep1 470,251,572378,466,223 28,217,448 25,763,514 11.83 13Pr2 rep2 515,334,056421,725,378 28,217,448 25,287,251 12.01 13Pr1 rep1 421,949,244340,966,300 28,217,448 25,733,843 10.58 13Pr1 rep2 383,229,986308,746,570 28,217,448 25,613,462 9.58 13Lg rep1 489,959,480 400,936,04628,217,448 25,342,333 11.59 13Lg rep2 505,568,682 415,435,288 28,217,44825,358,384 11.74 HPDE rep1 319,334,144 259,838,538 28,217,448 25,498,3388.14 HPDE rep2 409,472,974 329,823,993 28,217,448 25,755,008 10.07 38LgDMSO 381,562,458 313,237,618 28,217,448 25,509,209 9.67 rep1 38Lg DMSO462,809,566 378,726,614 28,217,448 25,716,790 11.67 rep2 38Lg 6AN668,822,798 546,162,307 28,217,448 25,987,966 16.62 rep1 38Lg 6AN408,683,276 329,596,955 28,217,448 25,586,266 10.20 rep2 A124PerMet388,402,336 313,722,746 28,217,448 25,551,642 7.39 A124Pr 348,071,524281,407,466 28,217,448 25,424,592 6.73 Normal 335,262,856 264,003,66528,217,448 25,423,437 6.37 A125LvMet2 299,092,468 241,196,683 28,217,44824,124,822 5.55 A125LvMet1 256,878,816 210,595,185 28,217,448 24,190,4595.09 A125Pr2 274,964,060 226,288,821 28,217,448 24,326,375 5.39 A125Pr1338,097,576 277,039,476 28,217,448 25,344,423 6.55 Total 7,989,272,699

Supplementary Data 1C: Summary of RNA-seq reads for all replicatesamples

Genes with Uniquely aligned at least Samples Total reads reads one read38Per rep1 136,981,522 126,989,515 24,629 38Per rep2 209,196,426194,285,575 25,767 38Per SFM rep1 131,806,836 120,661,977 23,750 38PerSFM rep2 170,418,406 157,947,694 25,204 38Lg rep1 157,009,238146,313,205 22,347 38Lg rep2 126,740,896 118,431,884 21,849 38Lg SFMrep1 157,587,660 144,483,739 23,085 38Lg SFM rep2 136,421,728124,124,770 22,387 38-Lv rep1 121,404,350 99,124,421 23,033 38-Lv rep2201,919,638 160,412,909 24,203 13Pr2 rep1 189,720,644 163,161,693 25,01113Pr2 rep2 154,397,366 132,746,732 24,122 13Pr1 rep1 194,020,194152,395,660 25,013 13Pr1 rep2 159,731,916 124,045,393 24,300 13Lg rep1258,937,646 222,764,245 25,530 13Lg rep2 104,375,982 89,301,099 24,908HPDE rep1 142,777,652 115,147,712 23,274 HPDE rep2 148,752,028119,731,612 23,348 38Per DMSO rep1 103,160,056 90,683,198 23,718 38PerDMSO rep2 205,086,938 175,208,405 25,285 38Per 6AN rep1 163,145,386144,907,391 24,514 38Per 6AN rep2 163,346,616 145,684,561 24,049 38LgDMSO rep1 183,580,116 158,065,524 23,609 38Lg DMSO rep2 177,808,970156,924,338 23,334 38Lg 6AN rep1 231,471,388 201,726,950 24,544 38Lg 6ANrep2 181,401,400 157,469,565 23,376 Total 3,742,739,767

Supplementary Data 1D: Summary of sequencing correlation coefficientsfor each replicate

Samples Modification Correlation coefficients between replicates 38PerK4Me3 0.8487501 38Per K36Me3 0.7908561 38Per K27Me3 0.8087827 38PerK9Me2 0.9533903 38Per K9Me3 0.973981 38Per K9Ac 0.8405352 38Per K27Ac0.9662306 38Per K16Ac 0.8958976 38Lg K4Me3 0.8977917 38Lg K36Me30.7457856 38Lg K27Me3 0.8283084 38Lg K9Me2 0.9345982 38Lg K9Me30.9870598 38Lg K9Ac 0.8557192 38Lg K27Ac 0.9749662 38Lg K16Ac 0.863448413Pr2 K27Ac 0.9795735 13Pr2 K27Me3 0.957889 13Pr2 K36Me3 0.970403 13Pr2K9Me2 0.9710885 13Pr2 K9Me3 0.9918936 13Pr1 K27Ac 0.9797524 13Pr1 K27Me30.9481441 13Pr1 K36Me3 0.9697859 13Pr1 K9Me2 0.9238059 13Pr1 K9Me30.9841044 13Pr1 K4Me3 0.9969566 13Lg K27Ac 0.9824737 13Lg K27Me30.9518218 13Lg K36Me3 0.9647369 13Lg K9Me2 0.9505661 13Lg K9Me30.9927826 HPDE K27Ac 0.9969341 HPDE K27Me3 0.9694532 HPDE K36Me30.9582686 HPDE K9Me2 0.9820727 HPDE K9Me3 0.9655677 HPDE K4Me3 0.99113438Lg_DMSO K27Ac 0.9523149 38Lg_DMSO K27Me3 0.9190836 38Lg_DMSO K36Me30.8830686 38Lg_6AN K27Ac 0.9515394 38Lg_6AN K27Me3 0.9328865 38Lg_6ANK36Me3 0.8750725 Average 0.9331653 Median 0.9556397

Supplementary Data 2. This provides summaries of chromatin domain callsfor LOCKs, large LOCKs, and ECDs for each sample including medianlengths, ranges, % genome coverage, and levels of individual histonemodifications in each type of domain. Median lengths, ranges, and %genome coverage for each individual heterochromatin modificationindividually (irrespective of domain location) is also included.

Supplementary Data 2A: Summary of large chromatin domains detected byChIP-seq

Samples Median K9Me2 K9Me3 K27Me3 (name, Ranges lengths Total length %of enrichment enrichment enrichment source) Domains Nos. (bp) (bp) (bp)genome (ΔRPKM) (ΔRPKM) (ΔRPKM) A38Per, LOCKs 2,648 100,001~ 311,2511,547,508,645 54.11% 0.082 0.003 0.102 Peritoneal 24,067,001 ECDs 2,02150,499~ 271,999 895,772,411 31.32% −0.116 −0.225 −0.139 8,760,499 Large344 500,501~ 1,287,251 589,313.343 20.61% 0.012 0.458 −0.020 LOCKs10,773,501 A38Lg, LOCKs 3,166 100,001~ 298,251 1,627,719,164 56.91%0.069 −0.101 0.118 Lung Met 14,074,501 ECDs 2,318 50,499~ 241,499838,469,352 29.32% −0.112 −0.243 −0.159 12,388,255 Large 226 505,501~1,340,751 416,070,225 14.55% 0.004 1.040 −0.152 LOCKs 10,428,001 A13Pr1,LOCKs 2,446 100,001~ 279,501 1,745,065,095 61.02% 0.038 0.063 0.124Primary 1 30,694,501 ECDs 2,008 50,499~ 255,167 914,964,363 31.99%−0.062 −0.176 −0.221 5,944,499 Large 77 504,001~ 994,501 97,292,5763.40% −0.045 0.640 0.043 LOCKs 5,223,501 A13Pr2, LOCKs 2,862 100,001~298,251 1,845,941,860 64.54% 0.065 0.051 0.107 Primary 2 24,749,001 ECDs1,968 50,499~ 277,249 914,565,095 31.98% −0.122 −0.141 −0.199 10,621,499GHDs 50 501,501~ 711,001 40,355,550 1.41% 0.015 0.990 −0.112 1,742,001A13Lg, LOCKs 3,140 100,001~ 293,501 2,052,192,139 71.75% 0.048 0.0270.089 Lung Met 31,395,001 ECDs 1,935 50,499~ 207,499 672,028,952 23.50%−0.125 −0.155 −0.231 3,447,581 Large 111 500,501~ 735,501 100,336,1113.51% −0.034 0.558 −0.180 LOCKs 2,878,501

Supplementary Data 2B: Summary of broad heterochromatin modificationsdetected by ChIP-seq

Samples Median (name, Large histone Ranges lengths Total length % ofsource) modifications Numbers (bp) (bp) (bp) genome A38Per, K9Me2 2,331100,001~ 335,001 1,449,305,831 50.68% Peritoneal 24,067,001 K9Me3 (large344 500,501~ 1,287,251 589,313,343 20.61% LOCKs) 10,773,501 K9me3(LOCKs) 711 102,001~ 219,501 207,546,709 7.26% 1,690,501 K27Me3 1,523100,001~ 186,001 360,026,522 12.59% 1,127,001 A38Lg, K9Me2 2,010100,001~ 255,501 979,113,009 34.23% Lung Met 14,074,501 K9Me3 (large 226505,501~ 1,340,751 416,070,225 14.55% LOCKs) 10,428,001 K9me3 (LOCKs)189 101,501 215,000 49,777,688 1.74% 1,027,001 K27Me3 2,649 100,001~244,501 882,095,148 30.84% 2,197,001 A13Pr1, K9Me2 1,696 100,001~176,501 407,098,196 14.23% Primary 1 1,428,501 K9Me3 (large 77 504,001~994,501 97,292,576 3.40% LOCKs) 5,223,501 K9me3 (LOCKs) 1,181 100,501~322,501 573,396,678 20.05% 11,051,001 K27Me3 2,211 100,001~ 232,001803,104,711 28.08% 30,694,501 A13Pr2, K9Me2 2,693 100,001~ 295,5011,738,982,692 60.80% Primary 2 24,749,001 K9Me3 (large 50 501,501~711,001 40,355,550 1.41% LOCKs) 1,742,001 K9me3 (LOCKs) 505 101,001~187,501 117,705,503 4.12% 1,203,001 K27Me3 1,828 100,001~ 220,001589,055,827 20.60% 2,887,501 A13Lg, K9Me2 3,091 100,001~ 283,5011,898,353,591 66.38% Lung Met 24,749,001 K9Me3 (large 111 500,501~735,501 100,336,111 3.51% LOCKs) 2,878,501 K9me3 (LOCKs) 711 101,501~194,501 168,847,710 5.90% 1,748,001 K27Me3 2,553 100,001~ 237,0011,003,794,552 35.10% 31,395,001

Supplementary Data 3. This provides all p-values calculated forsequencing experiments, as designated by the figure labels. Sensitivityanalyses for LOCK domain calls are also included.

Supplementary File 3A: p-values for H3K9Me2 reprogramming across LOCKs

FIG. 2A: H3K9Me2 Reduction Across LOCKs (vs A38Per) K9Me2 p-value MbK9Me2 Enrichment (RPKM < A38Per Reduction (vs p-value (Reduced Mb vsSamples (ARPKM) Wilcox Test) A38Per) A38Per, Chi-square Test) A38Per,Peritoneal 0.082 NA (reference) NA (reference) N/A (reference) A38Lg,Lung 0.069 p < 2.2e−16 763 Mb (52.7%) p < 2.2e−16 Metastasis A13Pr1,Primary 0.038 p < 2.2e−16 1110 Mb (76.6%) p < 2.2e−16 Tumor 1 A13Pr2,Primary 0.065 p < 2.2e−16 286 Mb (19.7%) p < 2.2e−16 Tumor 2 A13Lg, Lung0.048 p < 2.2e−16 204 (14.1%) p < 2.2e−16 Metastasis

Supplementary Data 3B: p-values for reprogrammed euchromatinmodifications from DE genes

FIG. 2E Genes Up-regulated from Euchromatin (vs Peritoneal: A38Per)pvalue K27Ac pvalue K36Me3 Sample (>A38Per Wilcox) (>A38Per Wilcox)A38Lg, Lung Metastasis p < 2.2e−16 p < 2.2e−16 A13Pr1, Primary Tumor 1 p< 2.2e−16 p < 2.2e−16 A13Pr2, Primary Tumor 2 p < 2.2e−16 p < 2.2e−16A13Lg, Lung Metastasis p < 2.2e−16 p < 2.2e−16 FIG. 2E GenesDown-regulated from Euchromatin (vs Peritoneal: A38Per) pvalue K27Acpvalue K36Me3 Sample (<A38Per Wilcox) (<A38Per Wilcox) A38Lg, LungMetastasis p = 0.0000007224 p = 6.41le−09 A13Pr1 p < 2.2e−16 p < 2.2e−16A13Pr2 p = 0.0000001401 p < 2.2e−16 A13Lg p < 2.2e−16 p < 2.2e−16

Supplementary Data 3C: p-values for reprogramming of H3K9Me3 acrossLOCKs

FIGS. 10A-10B: H3K9Me3 Enrichment over LOCKs Comparison H3K9Me2 (WilcoxTest) H3K9Me3 (Wilcox Test) A38Per vs A38Lg p < 2.2e−16 p < 2.2e−16A13Pr1 vs A13Pr2 p = 0.1086 p < 2.2e−16 A13Pr1 vs A13Lg p = 0.2364 p <2.2e−16

Supplementary Data 3D: p-values for reprogramming of modifications fromLOCK DE genes

FIG. 11A: DE genes Downregulated from LOCKs (vs A38Per) DNA K9Me2 K27me3K27ac K36me3 Methylation (>A38Per (>A38Per (<A38Per (<A38Per (<A38PerSample Wilcox) Wilcox) Wilcox) Wilcox) Wilcox) A38Lg p = 0.99 p <2.2e−16 p < 2.2e−16 p < 2.2e−16 p < 2.2e−16 A13Pr2 p < 2.2e−16 p <2.2e−16 p < 2.2e−16 p < 2.2e−16 p < 2.2e−16 A13Pr1 p < 2.2e−16 p <2.2e−16 p < 2.2e−16 p < 2.2e−16 p < 2.2e−16 A13Lg p < 2.2e−16 p <2.2e−16 p < 2.2e−16 p < 2.2e−16 p < 2.2e−16 FIG. 11B: DE genesUpregulated from LOCKs (vs A38Per) DNA K9Me2 K27me3 K27ac K36me3Methylation (<A38Per (<A38Per (>A38Per (>A38PerWi (>A38Per SampleWilcox) Wilcox) Wilcox) lcox) Wilcox) A38Lg p = 0.99 p < 2.2e−16 p =1.163e−06 p = 0.000000007195 p = 0.01699 A13Pr2 p < 2.2e−16 p < 2.2e−16p < 2.2e−16 p < 2.2e−16 p = 3.312e−08 A13Pr1 p = 0.51 p = 2.738e−06 p =1.362e−06 p = 0.0004268 p = 0.99 A13Lg p < 2.2e−16 p < 2.2e−16 p =2.362e−13 p < 2.2e−16 p < 2.2e−16

Supplementary Data 3E: p-values for reprogramming across Large LOCKdomains

FIGS. 13B-13C: Reprogramming Across Large LOCK Domains H3K9Me2 (>A38LgH3K9Me3 (<A38Lg DNA Methylation FIG. 13B Wilcox) Wilcox) (>A38Lg Wilcox)A38Per Large LOCKs p = 0.0006861 p < 2.2e−16 p = 0.000007037 H3K9Me2(>A38Per H3K9Me3 (<A38Per DNA Methylation FIG. 13C Wilcox) Wilcox)(>A38Per Wilcox) A38Lg Large LOCKs p = 0.00002502 p < 2.2e−16 p =3.52e−16

Supplementary Data 3F: p-values for 6AN RNA/ChIP-seq experiments

FIG. 19A: 6AN Down-regulation of DE Genes from LOCKs with Pre−ExistingRegulatory Modifications (6AN vs. other LOCK genes) K27Ac K36me3 K27Me3K9Me2 (6AN > Other) (6AN > Other) (6AN < Other) (6AN < Other) WilcoxTest p- p < 2.2e−16 p < 2.2e−16 p < 2.2e−16 p = 0.02238 value FIG. 19B:6AN Increase of H3K9Me2 Across Reprogrammed LOCKs (DMSO vs 6AN OverLOCKs Reprogrammed between A38Lg vs A38Per) K9Me2 K27Me3 (6AN > DMSO)(6AN > DMSO) Wilcox Test p- p = 2.29le−07 p = 0.6333 value FIGS.19C-19D: 6AN Decrease of H3K27Ac Over DE Genes Repressed From LOCKs FIG.19C K27Ac LOCK K27Ac Other K27Ac ECD Down Genes LOCK Genes Down Genes(6AN < DMSO) (6AN < DMSO) (6AN < DMSO) Wilcox Test p- p = 0.01549 p =0.94462 p = 0.8944 value FIG. 19D K27Me3 LOCK K27Me3 Other K27Me3 ECDDown Genes LOCK Genes Down Genes (6AN < DMSO) (6AN < DMSO) (6AN < DMSO)Wilcox Test p- p = 0.9417 p = 0.5139 p = 0.7957 value

Supplementary Data 3G: LOCK sensitivity analyses

Sensitivity Testing of LOCK Domain Calls Test Test Original Parameter 1Overlap Parameter 2 Overlap Parameters Called Analysis Called AnalysisCalled Domain Domain Percent Domain Percent Sample Lengths LengthsOverlap Lengths Overlap Small Peaks 5 Kb vs 5 Kb vs Removed 5 kb 2 kb 2Kb 10 kb 10 Kb 38Per K9me2 1449305831 1475751351 98.02% 138954331295.65% 38Lg K9me2 979113009 1042797151 93.44% 870263273 88.44% 38PerK27me3 360026522 383258588 92.44% 321712391 88.43% 38Lg K27me3 882095148886275657 99.53% 863791604 97.89% Minimal Merge 20Kb vs 20Kb vs Distance20Kb 15Kb 15Kb 25Kb 25Kb 38Per K9me2 1449305831 1387108909 95.42%1504614743 96.11% 38Lg K9me2 979113009 887961919 89.91% 105358404491.69% 38Per K27me3 360026522 314837912 86.64% 396029094 89.89% 38LgK27me3 882095148 848539120 96.00% 906322630 97.16%

Supplementary Data 4. This file lists all differentially expressed (DE)genes detected in each sample by RNA-seq, including level of expression,p-values, directional changes, and chromatin domains that each DE genemapped to. Analysis of recurrent DE genes detected across distantmetastatic samples and between control (DMSO) and 6AN treated cells isalso reported.

Supplementary Data 4A: Summary of DE genes between A38Per and A13Pr1detected by RNA-seq and mapped to chromatin domains (data notshown—publicly available on the World Wide Web atnature.com/ng/journal/v49/n3/full/ng.3753.html?foxtrotcallback=true#supplementary-informationby clicking link “Supplementary Table 7”).

Supplementary Data 4B: Summary of DE genes between A38Per and A13Pr2detected by RNA-seq and mapped to chromatin domains (data notshown—publicly available on the World Wide Web atnature.com/ng/journal/v49/n3/full/ng.3753.html?foxtrotcallback=true#supplementary-information by clicking link “Supplementary Table 7”).

Supplementary Data 4C: Summary of DE genes between A38Per and A13Lgdetected by RNA-seq and mapped to chromatin domains (data notshown—publicly available on the World Wide Web atnature.com/ng/journal/v49/n3/full/ng.3753.html?foxtrotcallback=true#supplementary-information by clicking link “Supplementary Table 7”).

Supplementary Data 4D: Summary of DE genes between A38Per and A38Lgdetected by RNA-seq and mapped to chromatin domains (data notshown—publicly available on the World Wide Web atnature.com/ng/journal/v49/n3/full/ng.3753.html?foxtrotcallback=true#supplementary-information by clicking link “Supplementary Table 7”).

Supplementary Data 4E: Summary of DE genes between A38Per and A38Lvdetected by RNA-seq (ChIP-seq not performed for chromatin domains) (datanot shown-publicly available on the World Wide Web atnature.com/ng/journal/v49/n3/full/ng.3753.html?foxtrotcallback=true#supplementary-information by clicking link “Supplementary Table 7”).

Supplementary Data 4F: Summary of DE genes recurrently up/down-regulatedacross primary tumor precursor (A13Pr1/2) and distant metastaticsubclones (A13Lg, A38Lg, A38Lg) vs. A38Per (data not shown—publiclyavailable on the World Wide Web at nature.com/ng/journal/v49/n3/full/ng.3753.html?foxtrotcallback=true#supplementary-information by clicking link “Supplementary Table 7”).

Supplementary Data 4G: Summary of DE genes between control (DMSO) and6AN treated A38Lg cells detected by RNA-seq and mapped to chromatindomains (data not shown—publicly available on the World Wide Web atnature.com/ng/journal/v49/n3/full/ng.3753.html?foxtrotcallback=true#supplementary-information by clicking link “Supplementary Table 7”).

Supplementary Data 4H: Comparison of overlaps between DE genes betweenmatched lung and peritoneal subclones (A38Lg vs. A38Per) and DE genesbetween control and 6AN treated A38Lg cells.

38Lg up/ Genes 38Lg down/ Genes 38Lg up/ Genes 38Lg down/ Genes 6AN down6AN up 6AN up 6AN down Coregulated/ GDF5OS Coregulated/ CCT6BCoregulated/ WDR25 Coregulated/ RP11- 6AN down: 6AN up: 6AN up: 6AN down400K9.4.1 1032/1968 915/2192 379/2192 277/1968 (52%) (42%) (17%) (14%)Coregulated/ EDN2 Coregulated/ JAK2 Coregulated/ TOMM7 Coregulated/C14orf105 38Lg up: 38Lg down: 38Lg up 38Lg down 1032/4368 915/4402379/4368 277/4402 (24%) (21%) (0.09%) (0.06%) C1QTNF2 TMEM63A IGFBP1 CCKCLDN2 FAM101A SLC9A3R2 RP11- 255B23.3.1 FAT2 KRCC1 C7orf60 CFTR(Coregulated: RBM24 ERRFI1 ZFYVE28 CD22 genes present in both 38Lg andmatched 6AN KMO LRRC6 CTSD RGS7 datasets) TPRG1-AS1 TNFAIP2 GATA6ERVMER34-1 DOCK2 MAST3 RP11- CCNI2 480A16.1.1 FAM101B ABCC10 GXYLT2ADAMTS14 KRT7 RP11- AC002472.8.1 ANO1 65J3.1.1 TCF7 SLC12A7 C3orf23PADI3 RP11- WASH7P TMEM43 CHST4 845M18.3.1 C4orf49 ANKRD13D FUT11 FER1L6SLC1A3 ZBTB7A IFFO1 TNNT2 DMC1 WASH6P SLC1A4 RP11- 597D13.9.1 TMSB15AIL32 DDT RP11- 6F2.4.1 LHX4 RNF217 IDI1 MYO5B KIAA1324 EFNB1 SARM1 KRT23ITPRIPL1 KHNYN HOTAIR MUC4 PPAPDC1A TMEM59 TSC22D1 RP11- 346D6.6.1 RGS5GAS8 NBAS ITGAM RP11- ABC7- ENG PTPRZ1 618G20.2.1 42389800N19.1.1RIPPLY1 RASSF7 SSTR5 GPR116 UHRF1 MRPS6 CRIP2 CYP24A1 SUSD5 RP11- NUDT18DHRS9 353B9.1.1 KRT80 GRAMD4 RPL31 GRPR KRT32 SERINC5 KIAA1143 AIF1LSLC26A7 TMEM102 DNAJC3-AS1 CGN TFPI2 TMPRSS5 C1R RP11- 314P12.3.1 RP11-TMEM63B GAB2 CYFIP2 314P12.2.1 MMP7 FAM109A FAM174B ATP6V0A4 KLHL23 TLR4SYT11 MPP7 C1orf110 C20orf96 DET1 KREMEN1 GRIN2A SMAD6 TBC1D8B TTN PADI2VAMP4 TAZ LEF1 AL162759.1.1 MYO1E UCP2 SP140 MYBL2 DVL1 RP4- SP6647C14.2.1 GLYATL2 AC069513.3.1 AARS ANXA10 RRM2 ITPKC DALRD3 NYAP2 E2F2USP18 SEMA3C MCOLN3 CPA4 RAB33B ATP2B1 DENND2A HHIP RP11- THTPA HSH2D325F22.3.1 MYPN ADHFE1 GPER C1orf106 RP11- LENG8 ITPK1 EVL 150O12.6.1CLSPN RILPL2 IL1RAP C7orf58 WDR69 TSNARE1 RPL29 RP1- 95L4.4.1 RP11-SPIRE2 EFHC1 IL11 150O12.1.1 AKAP12 MMAA CRTAP DDN DOCK10 SLC15A3 B3GAT1snoU13 PSG5 PLEKHM1 SEL1L SLC27A2 SPC25 RP11- ME1 DGAT2 403I13.8.1TM4SF4 STARD10 TBC1D9B SHANK2 FAM71D SNPH EEF1A1P5 HNRPCP OLFML2A EME2VKORC1 AC005083.1.1 OXCT1 DHRS3 SNX21 S1PR3 FAM111B MX1 KDELC1 ARL14snoU13 RRN3P1 AIP SAMD5 AC073130.1.1 FAM100A RP1- ARHGDIB 34B21.6.1CCNE2 DDO OSBPL6 GNG4 CDC25A TPRN C4orf34 CDC42BPG RP3- CD58 FAM18B2 SMO324O17.4.1 MCM10 PLCG2 TXNDC15 TIAM1 RP11- RP11- LIN7A NMU 424C20.2.1220I1.1.1 MSRB3 ZNF554 CTD- SOX6 2287O16.1.1 HEATR7B1 GLI4 FLYWCH1PPFIBP2 TP73 TRAF1 C11orf2 LY6D PRKDC ANKRA2 CHST12 PLAC1 DLEU2 SYNGR2FTX SYTL5 CTB- FAM113B CCNO SELL 164N12.1.1 SDPR CROCC AL844908.5.1MYO5C POLE2 HIVEP2 RP11- UNC5A 996F15.2.1 MEST RHBDD2 CPEB2 SPNS2 ASTN2GATS.1 PDIA6 BIK LAMP5 RALGPS1 STON1 PPYR1 MKI67 VWA1 ARRDC3 FAM46BTMEFF2 HEATR7A C7orf23 COL17A1 CPA5 HLA-K TESK1 MACC1 ANKRD18DP MAF1HEG1 DNAH12 ESCO2 TMEM8A RP11- CYP2J2 85K15.2.1 TUBA1B DDX58 MTHFD2LDLRAD3 WNT7A PYROXD1 RWDD2A RAB6B MCM4 CYP27C1 RPL10 BTBD11 AKAP5RNF145 PHACTR1 PLA2G7 TYMS ZDHHC14 NUDT22 TBC1D30 CDCA2 KLF7 TRIM4SNX25P1 CAP2 KYNU RHOG TESC RP11- LDLR EIF1B COL1A1 527N22.2.1 RP11-ZDHHC11B SSR4 FERMT1 297M9.2.1 DEPDC1B AC007383.3.1 ARG2 MED12L DPF1SRCRB4D TCEAL1 RP11- 582J16.5.1 RP11- LRP5L ZFPL1 BRI3BP 554I8.2.1 RP11-FOXC1 RGS10 LAD1 33N16.1.1 MAP3K14 AACS MEIS3 C16orf74 EXO1 SLC44A2LLGL1 CD163L1 RP11- SEPP1 C9orf37 ANXA8L1 253E3.3.1 FABP6 ARHGAP25 USE1AEN RP11- STAT6 YPEL1 C8orf46 184I16.2.1 FOSL1 RP11- PLA2G15 CDH127I1.2.1 CCDC99 VPS28 RPS27 XDH AC016831.7.1 R3HDM2 VEGFB XK FJX1C11orf35 CELSR3 ANXA8L2 SCN5A ARHGEF10L EPHX1 COL12A1 RP11- PSMD9 TINF2PPARGC1B 687M24.3.1 ILDR2 METTL7A ZNF70 C6orf132 MUC5AC TMTC2 C3orf18TTC3P1 AC092614.2.1 NFE2L1 PITPNC1 GALNT6 APOBEC3B C5orf13 ISL1 SEMA7ARP11- ZNF768 CORO6 F2RL1 54A9.1.1 NCAPG SIK1 NEK3 ALDH1A1 ATAD2 OSCP1RP11- CRABP2 313D6.4.1 SMC4 ACADS RPL27A CA2 FUT9 HERC6 RP11- FGFBP1216F19.1.1 ZNF488 CLDN9 ABCA3 SNAI2 TK1 RHEBL1 PITPNM2 PLS1 CTD- CHST6LGR5 KLHL13 2023N9.1.1 HELLS VMAC AGA RP5- 862P8.2.1 MMP24 AQP3 FAM113ARAB38 SLITRK3 DNAL4 GPR37 SLC7A8 ELOVL2 HBP1 VASH1 WNT3 SERINC2 ZG16BCAMKK1 SARDH AC002066.1.1 FAM100B MAST1 TPM1 DIAPH3 EREG RP11- RASEF574K11.20.1 RP11- ZNF862 RPL12 SKAP1 291L15.2.1 GINS1 NARFL NSUN5P2TRIM14 RP5- SWSAP1 RP11- CTSL2 968P14.2.1 477I4.3.1 DHFR YPEL5 NSUN5P1GK ROR1 SLFN5 HAUS4 VEPH1 MCM3 CCNDBP1 TMEM234 RP11- 800A3.4.1 RP1-PAQR8 MRC2 FGFR3 140K8.5.1 GLYATL1 SLC25A27 PYCR1 PADI1 CDCA7 GOLGA8BSLC26A6 CDS1 CSMD2 IKZF2 C3orf78 JPH1 RBPMS2 HSD17B11 CD68 FAM81A RBL1LMBR1L AP003068.23.1 GALNT12 RP11- KCNJ14 PAK3 DPY19L2 677M14.3.1 CCBE1DDR1 GMPPA ITGBL1 CCND1 C7orf53 EEF1A1P6 SYNPO2 E2F8 RELB AC093673.5.1RP11- 157P1.4.1 CDC45 WASH3P CBS ANKRD22 LMNB1 PDCD4 MIF4GD GRHL1C11orf41 NR1D1 CNPY4 KRT15 BEST3 NCOA7 GGT7 RP11- 93L9.1.1 CTPS GOLGA2LRP1B C15orf62 RASSF10 CEBPG FAIM2 AADAC POLA1 C2orf15 ZNF333AC112229.1.1 TUBB6 KLF13 AGXT2L2 TRIM59 HOMER1 GEMIN8 B9D1 DNAH10 CD200ARRDC2 EEF1A1 CTD- 2021J15.2.1 CENPM SLC39A11 ATRNL1 KRT19 ERCC6L SAT2PAOX KIAA1199 HMMR ZER1 CTSA GPM6A CENPW KIAA1407 SLC45A1 AKAP6 RP11-OAS1 RAB3IL1 GPR110 298I3.4.1 RP11- AC004410.1 C6orf1 QPCT 259P6.1.1THBS1 DGAT1 CTD- RP11- 2314G24.2.1 382A18.1.1 GPR63 RNF103 CYP4X1METTL7B DTL EFHC2 HCFC1R1 PHACTR2 RP11- C17orf69 FAM70B PLEKHA7101K10.8.1 RP11- LINC00493 SHMT2 PLAU 204J18.3.1 WDR4 ST6GALNAC2 SEC14L5SLC4A3 BRIP1 KLHL31 CLK4 KRT18 LMNB2 LGMN UGDH-AS1 C3orf52 C14orf49LLGL2 ITGA11 SYTL2 EIF6 ENDOV CGREF1 PKDCC ARHGEF26 CST1 RPL22L1 ASB9ETNK2 RP13- FKBP2 SLC22A20 516M14.1.1 POLQ LIPA OXA1L IGSF3 RP11- FAM13AC10orf102 CLDN10 573I11.2.1 APLN ST3GAL1 DNASE1L1 MBOAT1 RP11- APOBEC3GHIGD2A TMEM169 304F15.3.1 NDC80 PLAUR MLLT3 SYK MCM6 ACYP2 AP001496.1MAST4 CAV1 RTN2 FAM156B SDC1 RP11- HIF1A TRPT1 RP11- 53M11.3.1613M10.6.1 MARK1 DNASE2 RP11- PPM1H 571M6.6.1 CTA- CA11 RP11- GCHFR445C9.15.1 243J18.3.1 USP13 SLC7A7 SGSH RHOD RP5- ABCA10 CTD- EZR1172A22.1.1 3074O7.5.1 UCN2 PTK6 CNTNAP1 RP11- 303E16.2.1 MCAM RPS6KA2TNFRSF10C RCC2 ZWINT WHAMMP3 B3GNT1 KDR LINC00460 ENGASE LRP1 RP11-416I2.1.1 BIRC5 LPCAT4 P2RX6 GPR65 FLNC EPS8L3 SSBP2 BCL7A SYNE2TMEM198B ZC3H6 PPCDC HMGN2 PRICKLE3 CCNG1 NBPF10 ADCY3 PDE4DIP TBC1D4AC108463.1 PDSS1 GPR108 MAGED2 CADM4 CDCA3 TMC4 DNAJC1 SPTBN5 AXL PTP4A3PCK2 CUEDC1 KIAA0101 ITGAX MANF CMTM4 NAV3 TSSC4 ZCWPW2 CDK5R1 KIF11ANO9 C1orf213 C19orf21 LPCAT2 PPP1R3B CACNG6 C1orf116 KIF4A ANTXR2AC004540.5.1 UNC5C CTD- ARHGEF3 TSLP ALDH5A1 2334D19.1.1 EML5 C10orf32RP5- MB21D2 1103G7.4.1 NME1 PGM2L1 C1S RP11- 7K24.3.1 ARHGAP11A HLA-HSPATA25 DSG2 MCM2 TUBG2 PPAP2A PLD6 SLC5A11 SLC16A3 RP11- GALNT5390P2.4.1 CHN1 CCNL2 RP11- ZNF185 539L10.3.1 KIF23 SGK1 SH2B2 P2RY2ARHGAP19 MEF2C NEURL2 PLCXD1 SLC8A1 CAMTA2 KLHDC2 AIG1 FBN2 HLA-F-AS1SHISA2 PALM FKBP5 PIWIL4 PXK PKP4 IGSF9B LSS WBSCR27 ZNF462 KIF15 PCMTD2DSEL SAA1 NUP210 CFI TRIM46 SPATA13 AS3MT ARL4D CTSF LAMA3 TNFAIP8L3 MVKKLHL35 NID1 PM20D2 CDH17 PDIA5 PEAR1 OPN3 MAFF PLK3 PYGL RRM1 CACNB1ZNF815 USP40 DNA2 C2orf63 NICN1 DIS3L DEPDC1 IL15 SCAND1 ID2 PDE1AST3GAL6 CALHM3 FUT1 ALYREF C9orf7 RP5- PRSS3 827C21.4.1 MORC4 ENDOULETMD1 NMNAT2 FBXO5 PIM3 JAKMIP3 DNAJA4 DPY19L2P1 CLDN7 LZTFL1 DOCK9CENPF DAB2IP CTNS DDI2 GPR19 TARSL2 RP11- PLAC8 280F2.2.1 SYCE2 REC8PDK1 FBP1 GINS4 FAM160A2 MBL1P RAG1 XRCC2 ZFP36 LHPP PRPF4 DARS2 RP11-BLVRB RPS6KA1 73K9.2.1 HMGB3 STARD4 CALHM2 DSP KIAA1524 SGK2 RP11-SLC22A5 277P12.20.1 NUP188 CIR1 PTCHD2 AADAT DPF3 MAPK8IP3 UST FARP2STMN1 USP20 IRF2BPL MCC WDHD1 RP11- PLK1S1 ZNF658 149I23.3.1 NCAPD3RP11- TTC39B B3GNT5 108M9.4.1 CCNE1 HIP1R ABHD14B NT5DC3 TRPV2 CYP4F3DLG4 ABCC9 CDKN3 SH3YL1 PCDHGA7 PNPLA4 PRR11 TNFSF12 MAGED1 ZNF717 CSRP2TMEM8B LEPREL2 RAVER2 COTL1 IGSF8 LOXL2 MREG MSH2 PTK2B SLC43A2 RBP1KIF18B NR4A2 ZEB1-AS1 RAPGEF3 SKP2 SPIRE1 LENG8-AS1 CNKSR3 ADORA2B DLL1C17orf72 RP11- 757F18.5.1 GFRA3 TMED1 C16orf93 PRSS12 ZNF724P SQLEARHGAP4 HOOK1 RASGRF1 ABCC6 CNTD1 COL16A1 FAM83D VSIG10L PCDHGA3 MST4.1BUB1 FA2H STK32A CMIP MTHFD1 OSBPL7 SLC29A4 SMC5 DHX9 KCNIP3 LEPRE1ANXA2P2 NRG1 LYNX1 HOMER3 STEAP2 PCNA ULK3 MCEE NUDT14 UTP20 HINT3 XBP1MMD MIR17HG FDFT1 ALDH1L2 ANKRD56 ANLN TRIB1 MAP2 SURF2 RUVBL1 NPDC1NNMT SH2D3A MYO1B VEGFC NUCB1 MAP3K1 NCAPG2 GPRC5C KLRC2 UACA FH SIGIRRTMEM158 TAF2 GNG11 TCEANC SEPT7L DCBLD2 GS1- MKNK2 RP11- RP11-465N13.1.1 161M6.4.1 295M3.1.1 MT1L N4BP2L1 AC079922.3.1 NAALADL2 TNS1KCNMB4 PCDHB7 CNTRL BARD1 RP5- HSD17B7P2 CD82 1182A14.3.1 THOP1 GET4NAT6 FBXL19-AS1 SKA1 FKBP10 PTX3 RHBDL2 C1QL1 RDH10 ZNF836 RP11-448G15.3.1 C11orf82 SLC22A15 TMEM106A RP11- 357H14.19.1 CCNA2 CCDC57TFF2 TUBGCP5 SLC35F3 SHB CYP1A1 INADL C10orf140 TMPRSS3 PPP1R3E C9orf64NEXN NYNRIN TMEM120A ARAP2 ASF1B TNFRSF9 POLN KRT16 PRTFDC1 SESN3RAPGEF4 ALS2CL FAT3 A2LD1 RABAC1 H2AFX RP11- CACNA1G 475N22.4.1 PRIM1ZNF628 LOX SNX18P3 JAG2 FAM151B RP11- RP11- PTPRM 973F15.1.1 244H3.1.1ADRB2 GOLGA8A CLEC2B FBXL13 TP53INP2 KDELR3 CDC20 WASH4P ISYNA1 CENPKTNIP1 RASL11A CDCA4 RP4- CALR 659J6.2.1 ATAD5 CCDC126 RP11- 108K14.4.1SMC1A PTPRB METTL12 FAM196B U6 BNIP3 UGT1A1 SPRY3 FAM175A CTD- MMP15CRLF2 2574D22.2.1 MCM5 SLC23A3 TMEM231 MELK TBC1D3F CRELD2 LYPD1 ABCA5PLXNA3 DLGAP5 RP4- AC026202.3.1 798A10.2.1 NUSAP1 CLDN15 ASNS TUBBP1TMC7 RASIP1 MYH10 TCTEX1D2 SFRP5 MIR155HG NOS3 RP11- 66N24.3.1 CCDC138ARHGEF16 ABCC3 ERCC2 TPBG RIBC1 KIF20A FAAH HSP90B1 TMEM14B TMEM150AHYOU1 H2AFY2 LRRC56 BAMBI OXTR CEP85L TPP1 RDM1 MIA ZCCHC24 PLEK2 CDH6FAM161B SHCBP1 PON3 AC004080.12.1 GJC1 CCDC92 MAPT SNRNP25 PTPRHAC018755.11.1 KCNQ5 FOXQ1 PLOD2 ODC1 J01415.23 MAN1A1 EBNA1BP2 PPFIA3NUCB2 FABP3 SMPDL3A DERL3 MCM7 AC007283.5.1 SCN1B CHAC2 ALPPL2 FN1 BORAMTHFR PLCD1 ASRGL1 WASH2P CCDC85B VIPR1 RSAD2 BTBD19 PTTG1 LIMA1AP000769.1 ACTB PPP1R16A AC147651.3.1 TOP2A PIK3C2B ATHL1 NEIL3 XAF1CYP2E1 ALDH1B1 KIAA0513 FAM182B CEP250 GRAMD1C PDIA4 FAM131B GLDN ZFP2FAH GPCPD1 SLC25A29 CIT CABP4 RAB24 CDCA8 NXNL2 RP11- 755F10.1.1 CDK2SEMA4C QPCTL EZH2 PHYHIP ALDOC PSMC3 AC093734.11.1 AKR1C1 FGGY IRF9LRRC29 NXPH4 ZNF517 RP11- 307O13.1.1 FRMD4A RP5- SEMA3F 1187M17.10.1CSPG4 HIST1H1C RUSC1-AS1 CSE1L ANKRD42 EVI2B KIF20B ZC3H12A TIE1 C2CD3JUND HSPA5 PPIAP29 SEMA4B TSPYL2 CTA- CTAGE5 HERPUD1 221G9.10.1 TPX2NLRP1 AQP2 DKK1 GNE RP4- 794H19.2.1 PFAS LTB4R2 C2orf16 PBK BBS12 AKR1C2TRIP13 DICER1-AS ANGPTL4 CCDC18 CMPK2 HCN2 UBE2T CALB2 NOG AL357673.1DHRS2 AKR1B1 PDE12 LRFN3 ST7-AS1 NUP155 PROC EVI2A ARNTL2 PROS1AC022007.5.1 POLD2 TMEM80 ZNF575 NCAPD2 RALGDS RCN3 VCL SLC6A8 PCDHB15CCDC85C TMC6 RP13- 895J2.7.1 FANCD2 HMGCR C9orf150 RP11- TBC1D17 ANGPT1117P22.1.1 MYLK TBC1D3 ARSA RP11- GRN RP11- 394B2.4.1 49I11.1.1 PRLRROM1 PRPH AP2B1 MID1IP1 AC004383.5.1 RASSF2 C15orf61 P4HA1 PRKAG2SPATA20 ANGPTL2 COL4A6 FAM78A AC002480.4.1 PSMC3IP STAT2 MTMR9LP ARRB2PYGM CRELD1 CAMK4 RHBDF1 UPB1 TSPAN2 LRCH4 AC002480.3.1 RFC2 PODN RP11-554A11.9.1 PLK2 SPRY4 GALNT9 SRRT CITED4 AL137145.2 WNT7B LIPG VLDLRADORA1 CAPS IFITM10 C22orf29 EIF1 LDHD HTR1B CYP4F12 ERO1LB ITGB8AKAP17A NTNG2 ZNF660 ACTR1B NPY1R EPB41L2 WDR66 COQ3 AP001468.1 AASSAP001372.2.1 PAICS FAM214A CENPI GSN CASC5 FAM193B TUBA1C TPRG1L NUDCD1ARHGEF2 L2HGDH TNFAIP3 CHML CDA CCNB1 PLA2R1 RP11- ZSWIM4 799O21.1.1DNMT1 IRAK2 CDK1 LYZ RP11- TJP3 673C5.1.1 NUP37 C8orf55 TPMT CTB-131B5.5.1 AURKA AC103810.1 PEA15 PCDHGB2 NRGN KLC4 PLK1 RP4- 541C22.5.1ZNF124 SIX5 CGNL1 SEL1L3 SNRPD1 MIR29C PTPN14 MZF1 CDC7 ADAM8 DERA WDR45PYGO1 ZNFX1-AS1 LRRCC1 AC017099.3.1 FAM173B ARSD AHCY DLX4 UBAC2-AS1 HK2HIGD1A HS3ST1 GMNN ABCG1 NCAPH CCDC146 CEP128 EGLN3 SLC38A5 CLK1 TUBB4BUPK3B RP11- SLC16A6 512F24.1.1 BICC1 HLA-F HMGB1P5 NT5M NUF2 BTG1 VCANC7orf63 FAM64A RASSF9 DNAJC9 CTSL1 RANBP1 ENDOD1 C4BPB FAM116B C9orf140KNDC1 SKA2 PPP1R3F RP11- LARP6 181C3.2.1 PKMYT1 FBXO6 RFWD3 CCDC69 TCOF1PRRT1 KNTC1 CTD- 2258A20.4.1 DKC1 TSPAN1 MPP2 RP11- 362F19.1.1 CCNFSLC2A10 C1orf112 CTD- 2341M24.1.1 AC046143.7.1 C2orf81 STMN3 FZD4 OIP5RAB40C DGKH EXD3 NRM IFIT3 RFC3 PCDHGC5 RBBP8 RP11- 285F7.2.1 HTR7P1RHBDF2 NMT2 PRICKLE4 ARHGAP11B RABL2A DPYSL3 WBP1 TCAM1.1 LAMB3 YBX1RP4- 697K14.7.1 HSP90AA1 TMEM91 ACTA2 PCDHAC1 MPP5 TRIM2 C8orf84 C16orf7ASPM RTP4 LRRC8C KIAA1875 TBC1D7 RP11- 540D14.6.1 RP11- JUP 462L8.1.1ZNF367 NEU1 RP11- DDX60 956J14.1.1 CCT5 HOOK2 C6orf52 BCO2 NEK6 TBC1D8TSPY26P PRRX1 MRPL1 KRTCAP3 FARSB PINK1 PKP2 NFIL3 CAND2 LCN2 MRTO4 TYMPKIAA0586 RP11- 429J17.2.1 DEK WARS FAM54A COL6A1 FEN1 LZTS2 RP3- NPIPL2510D11.2.1 SMC2 LCA5L RELT KB-1460A1.5.1 SGOL1 CYP4V2 ANKRD1 FAM59ASUV39H2 SEZ6L2 KDELC2 PTPRE HMGB2 NR2F6 ATRIP COL11A2 SACS RENBP DEPDC7AQP6 HMGB1 ULBP1 RP11- CX3CL1 380J14.1.1 POP1 CCDC149 GFAP ASAH1 NUP205TNFRSF14 CENPA C12orf63 TPGS2 C12orf57 CD109 PSPN WDR3 RP11- 263K19.6.1NOP56 MAPRE3 PTRF RP11- 44N21.1.1 PHIP TNFSF13B QSER1 FMO5 FAM86A PRXPOC1A GSDMB CYB5RL JUNB H2AFZ HMGCS1 AC092329.1 CSF2RA RAD51AP1 HEXDCCCP110 PLXND1 CEP55 NFAM1 ZNF347 PER1 NCS1 CALCOCO1 RAD54L DRAM1 MDM2AOC2 SMTN GAS7 CDC25C IFIT2 TFDP1 MTRNR2L9 NEURL1B TSTD1 TBCD NR4A1KRBOX1 TM6SF1 SAE1 CBLB CHEK1 CCDC24 RP11- FAM66C 678B3.2.1 REV3LC9orf16 SPC24 KDM6B TMPO TBX6 DHRS4L2 MMP28 MYOF ACSL1 TUBA4A G0S2 ADAT2CDC42EP2 RP11- SCD 348A11.4.1 BST1 NFKBIA FOXM1 PLEKHF1 AC027612.6.1AGFG2 RP11- LEPREL4 1334A24.4.1 UBE2N ULK1 PRIM2 ADAMTS13 S100A2 CAMK2N1NEGR1 PDE7B HNRNPAB AC073343.1 CCDC41 AC002117.1.1 CAMK1 SPSB3 GTSE1SLC5A12 MMP2 PLA2G6 BOLA3 HOXD4 SYT15 HS3ST3B1 BCL2 JAK3 ECHDC3 C10orf10KIFC1 NFKBIZ G3BP1 DUSP8 PFN1 NEIL1 RAET1K ECEL1P2 MNS1 RP11-783K16.13.1 SIRPA KLHDC1 RP11- MST1 152N13.12.1 PORCN ROBO4 NOC3L SYT17RAD18 NR1H3 ESPL1 GDPD3 PTER CEBPB RP11- SDCBP2 1277A3.2.1 NUP107 NOVSGOL2 DNAH7 DIO2 IER5L ZNF726 FANK1 SFXN2 ORAI3 FRMD6 SLC5A3 CENPNB3GALT4 NT5DC2 ZDHHC1 RFC4 KIF27 NOP16 GIMAP2 TUBG1 CDC42EP5 DZIP1AC006028.9.1 IPO5 PRSS16 LHFP TGFBR3 ARHGAP22 FASN TMEM48 ATP1B1 RPA3C17orf108 TMCO7 EPHB6 THSD1 BCL6 AC013461.1.1 RP4- 811H24.6.1 CCDC165RP11- 496I9.1.1 MAGOHB CXCL2 EFEMP1 CACNA2D4 EED PPM1K KIF14 C17orf103NUP35 MXD1 DTYMK MAGIX SSX2IP PTPRCAP CELF2 DPM3 NETO2 EVPL PHLPP2SLC2A13 PTBP1 IL1B FKBP3 KCNE4 URB2 INPP5J EEF1E1 FRAT1 BEGAIN DUOX2CACYBP FBXL15 HMGN5 C15orf48 HJURP FAM86FP DBF4 NR3C2 XRCC3 STX1B TMCC3RP11- 273G15.2.1 SPAG5 RASSF4 AC108488.3.1 P2RX4 ATOX1 TPD52L1 SLC25A3RP11- 420G6.4.1 TUBB P2RY11 CCDC152 ADCK3 CENPL PARP10 GAS6 AC103810.2RP11- HERC5 540A21.2.1 NXT2 TFEB LRR1 FAM84B E2F1 AC093627.10.1 RP11-OAS2 122A3.2.1 KCNH4 ARHGAP9 MYEOV FBXO24 TMEM194B CD34 RANGAP1 CD14CNTF OTUD1 NASP ICA1 MAP7D3 AC005152.2.1 RP1- WDFY3-AS2 239B22.1.1 DUS2LHIST1H2AC FKBP1A SELPLG NOLC1 PDGFRB ZNF702P RP11- 566K11.1.1 CDCA5 SHC2IMMP2L CES3 SUPT16H GBP5 MIR621 MYL5 RP11- JHDM1D 64D22.2.1 CEP97 YPEL2ITGB3BP DHX58 ERCC8 IFIT1 WDR62 RP3- 395M20.8.1 ZNF239 DFNB31 RP11- LYG1680F8.1.1 GNL3L FAM66D MAPKAPK3 RP5- 882C2.2.1 ADK IL1R1 CENPV LTB4RUTRN PDXDC2P PSMD1 IL4I1 GTF2H3 FOXO4 USP49 AC127496.1 C3orf26 ANKZF1CYP26B1 CSF2 JAM3 LENG9 SPA17 AC097500.2.1 BTG3 CCDC19 MRPL15 FURINKPNA2 IFIH1 RRP15 GBP4 HSPB11 CCNG2 C17orf89 FZD1 HAT1 SNHG5 UBE2S PDZD7RBM12B DAPK2 MRPL47 C20orf195 MSI2 C9orf163 KIAA0020 PCSK4 PTPN1 KIF26BGLRX3 SERPINE2 CFL1 MSMO1 CDH24 CTC- 523E23.1.1 HNRNPA3 HIST1H3E XYLBPAQR6 DRAP1 P4HA2 ACTR3B AOC3 CHCHD3 IRS2 H2AFV MAFB NSMCE1 MXD4 COLQDNER CEP41 MDGA1 NUTF2 CH25H SMARCC1 TST KIF2C RARRES3 ATP5G1 UNC13AISPD MST1P9 CHTF8 DNAH2 IPO9 KCNK5 AC026271.4.1 PPIL6 TTF2 RP11-1391J7.1.1 DHRS4 RHPN1 AC068282.3.1 FOSB SF3B3 RP11- 202P11.1.1 GSG2MIR29B2 AHCTF1 CEACAM1 LPHN3 RNF24 KIF22 YPEL3 IMMP1L GPT SNRPB CALML6DCLRE1A RP1-163M9.6.1 ZNF681 NPPA-AS1 CDH2 PELI2 GPR125 FAM71E1 ANKRD18ABPI RP11- RASSF5 110I1.12.1 SCD5 TMEM53 ARMC10 ABTB1 AC009948.5.1 UCNKIF5C IDUA RP11- GDPD1 381E24.1.1 PACSIN3 RP4- 758J18.10.1 GNB4 BMFRP11- TRIB2 58E21.3.1 PARP1 DYRK1B HSPA4L SCNN1D PRPS1 CLIP2 PPIL1 INHAXRCC6 FAM47E COQ2 LRRC24 CBX2 GFI1 LBH EPB41L4A-AS1 RNASEH1 MVD RFT1THBS3 C1orf114 CARNS1 ITSN1 C16orf79 ATXN10 SLC16A13 WHSC1 CTD-2292P10.4.1 RP5- PDE5A 991G20.4.1 SAAL1 CTC- 378H22.2.1 FER MUC1 RP1-AC008440.10.1 152L7.5.1 MCM8 PCSK9 C4orf10 CTD- 2547L24.3.1 CROT ICAM5GTF2H2 MUC20 BAG2 PPP1R3C METTL8 KLF4 SEC14L2 NINJ1 C20orf94 CLIP3 HDAC8CTD- 2517M22.14.1 PRMT3 AC022098.1 HAUS1 ABCA6 POLR3G BBC3 CCT2 RP13-15E13.1.1 RP11- CXCL3 14N7.2.1 AC034193.5.1 RP11- 369J21.5.1 CNTLN PRR15CCDC34 C14orf45 DLX1 TMEM198 ABCC4 ZNF425 RFC5 GAL3ST1 PSMC5 SLC1A7CSTF2 ISG15 LIG1 ATF3 SLC19A1 ICOSLG BRCC3 ATP8A1 ZW10 LINC00324 AFAP1L1FBXO32 CDC123 EXOC3L4 RP11- SRCIN1 521B24.3.1 LRRC58 HSF4 ALMS1 DBPFAM111A CDH3 PUS7 AC021593.1 DYNC1H1 RNF152 EFNB2 ISG20 BCAS4 KIAA1683MYH9 RP5- 885L7.10.1 HSPA14 CARD14 TMEM56 PIP5K1B ZDHHC2 NCF2 KIF18APPP1R32 CDK4 RAB17 CKAP2L CHPF MRE11A KLHL24 PSRC1 PDGFRA NUP88 DNAH10OSRCC1 RP11- 454H13.6.1 GEMIN4 ABCA7 C1orf74 TCP11L2 VRK1 S1PR1 PSMC1HIST1H2BD DOCK1 NEURL3 ALDH3A1 SYT5 GLT8D2 GPR35 KATNAL1 CARD9 SERBP1CTD- 2313N18.5.1 WDR77 LIPH PHTF2 EFNA3 PSMB2 C3 BCAT1 MID2 STRA13 RP11-536G4.2.1 UBE2C PNPLA7 PHEX CYP7A1 ADSL TESK2 HSDL2 TNFSF15 CEP192ZNF385C GOT2 RP11- 757G1.6.1 STIP1 FBXO2 VBP1 LRRN1 GNPNAT1 ZSCAN4 DLC1PRSS27 FUBP1 EFNA1 HEATR1 CNNM1 TBC1D1 DDIT4 ECT2 SCN9A GALNT1 C2 EXOGCCDC114 CTD- RRM2P3 2366F13.1.1 POLA2 AP000696.2.1 SLC25A15 PDE4C NEU3CATSPERG DOCK5 ELF5 CETN3 HS3ST3A1 BMP4 AC073321.5.1 CCDC72 CFB SORL1PARM1 UCHL5 C1orf145 ARPC4 C19orf51 FAM198B CTC- 454M9.1.1 KIAA1586CCPG1 GSTO1 CTH AC097359.1 MALAT1 BMP2K TRIB3 SSBP1 NYAP1 C3orf67 KLF9SMC3 LINC00176 AP1B1 IER3 ACAD9 GBP2 C2CD4C VEGFA SLIRP SAT1 SNAPC1BAIAP3 RPL26L1 RP11- 93B14.5.1 MGAT5B ANKRD24 FSTL1 GAA PARVB CASP5TIMM23 OASL KLF12 MLXIPL C1QBP RP11- 115C10.1.1 CCRL1 TSC22D3 SLC16A7OPRL1 CDC6 SERPINA5 AC004381.6.1 SLCO4A1 CBFB IGSF10 APAF1 S100P PRKAR1BEPAS1 FAM72D SCARF1 FOXRED2 TNFSF10 TMEM209 ECT2L CEP76 PSD DLG5 CHAC1AC003665.1.1 ANG ACTL6A CA9 PDP1 PIK3IP1 C11orf51 FLRT3 RPL39L RP11-736K20.5.1 NUBPL CEACAM22P SFPQ MYO15B EME1 DPEP1 FANCM KLK10 HMBS ADM2HNRNPR ACCN3 HDAC9 LDB3 HECW1 FER1L4 GCSH CEBPD LAPTM4B TMEM105 GDF11CCDC40 NAV2 RP3- 395C13.1.1 NR2F2 ADCY4 INO80C NKPD1 PPAT SLC6A9 UMPSC13orf33 CACNB4 SERPINA3 PLCL2 ACSS2 PRMT5 SLC2A6 ACADSB EGR1 INCENPTSPEAR-AS1 LARS2 MAPK15 ZNF714 RP11- 178D12.1.1 CCDC86 FOS PSMD2 ANGPTL1LRRC20 ZNF467 ROCK2 RP11- 712B9.2.1 C12orf48 PRSS35 TNPO1 NHLRC4 PPAP2BSLC6A12 C14orf126 ICAM1 CDC42BPA C2CD4A BCCIP GPR132 PSIP1 STRC LSM3C6orf223 MALT1 NFE2 AAK1 CTD- 2319I12.1.1 CABYR FAM167B RBM12 PNCK CYCSTPPP3 SFMBT1 ITGA10 MPHOSPH9 ODF3B SEH1L WFDC10B DCAF13 GRIP2 TIMM10TXNIP LYRM1 NUPR1 FAM171A1 ARRDC4 CENPH SSPO RP11- TBX4 117L6.1.1 BRIX1LAMP3 PEG10 SLCO4C1 THOC7 HPN CDCA7L FAM132A HPRT1 GAL3ST2 CEP170 NGFRSF3A3 C2CD4B PMVK RANBP3L HSPD1 C17orf28 TDP1 C21orf90 DNMT3B MYO15ACKAP2 LRRC4C C12orf24 PLCH2 ANKRD28 HSPA6 CKAP5 IGFALS POLR1E KLBDCUN1D5 AC005013.5.1 ORC1 CSF3R KCTD1 RYR1 PSMA7 CCDC134 PSMC2 PLS3 PIGNCTD- 2224J9.2.1 NUP153 ME3 PITPNM3 ABCD3 IQCC DCLRE1B USP1 AC108463.2.1UPF2 SSRP1 ALG8 STARD13 NEK2 FCF1 GNG12 MASTL TBC1D5 AP3B1 CBX3 PGRMC1ATG3 POLH PDCD11 RP11- 666A20.1.1 ZDHHC23 RGS17 FAM72A SEPHS1 FAM122BVOPP1 PSMA3 FLVCR2.1 LIN9 MANEA FAM208B GTPBP4 TTI1 CCDC88A FAM48ATMSB15B SELRC1 RIMKLB WDR17 ODZ3 CNOT1 SMCHD1 RDX POLD3 LAS1L CLTCPPP1R14B GEMIN5 AGK C10orf125 SMS PDAP1 LYPD6 RP11- 290F20.1.1 SLC36A1TRAIP RP6- 65G23.3.1 PPIA RP11- 85G18.4.1 KCNC4 GDAP1 CABLES1 LGALS1RPP30 PI4K2B DSN1 ZNF620 USP6NL C9orf100 CAV2 PTCD2 KIAA1147 TOP2BTXNRD1 MYL6 RNASEH2A RBFOX2 CCT6A AMMECR1 DLD BAI2 ORC5 TIMM8A BEND6TSC22D2 NAP1L5 ATIC SIGLEC15 AP4S1 BAX TTK WDR12 TFAM GPRIN1 PRDX1GEMIN6 MMACHC JUN ERC1 EPHB2 XPO6 KIF24 SRGAP1 AURKB TRRAP GPD2 SCFD2SMAP2 GSS

Supplementary Data 41: Comparison of overlaps between recurrent DE genesacross all samples (vs. A38Per) and DMSO vs. 6AN treated A38Lg cells

Recurrent Up/ Genes Recurrent Down/ Genes Recurrent Up/ Genes RecurrentDown/ Genes 6AN Down 6AN Up 6AN Up 6AN Down Coregulated/ PPIACoregulated/ SAT1 Coregulated/ EEF1A1 Coregulated/ KRT19 + 6AN down: 6ANup: 6AN up: 6AN down: M2:M52 255/1968 332/2192 82/2192 111/1968 (13%)(15%) (0.04%) (0.06%) Coregulated/ DYNC1H1 Coregulated/ ATP1B1Coregulated/ AKR1C2 Coregulated/ DCBLD2 Recurrent up: Recurrent down:Recurrent up: Recurrent down: 255/891 332/1842 82/891 111/1842 (28.6%)(18%) (9%) (0.06%) CCT5 GRN RPL12 PLAU YBX1 JUP RPL31 EZR HNRNPA3 LCN2RPL29 GPR110 XRCC6 CST1 IDI1 COL17A1 PRDX1 TXNIP XBP1 ANO1 SERBP1 LAMB3ATP2B1 PADI1 DPYSL3 DDR1 HERPUD1 SDC1 ECT2 DUOX2 MTHFD2 CD82 CBX3SLCO4A1 MAN1A1 CGN SLC25A3 GSN SHMT2 LY6D HMGN2 LIMA1 MAP2 CRABP2 TMPOLYZ ABCA3 DOCK9 CYCS MUC1 TOMM7 SPNS2 PAICS GNE ASNS RAPGEF3 MYL6 DAB2IPPYCR1 FGFBP1 NOP56 TNIP1 TBC1D4 RBP1 HNRNPR LIPH UCP2 PRSS12 FKBP1ARHBDF2 CELSR3 C1orf106 THBS1 HS3ST1 LETMD1 QPCT DEK RDH10 KLHDC2 STEAP2ASPM MUC20 ARG2 ARHGDIB NUP210 CLIP2 LEPRE1 ZNF185 PCNA KLK10 SLC25A29DHRS9 HNRNPAB SLC44A2 PCK2 CDC42BPG ATIC EFNB1 SLC26A6 NBPF10 NCAPD2NFKBIA PTPRM MUC4 RANGAP1 EVPL SYT11 PLEKHA7 MYO1B ENDOD1 FAM113A AIG1MTHFD1 ICAM1 GAB2 RP11- 800A3.4.1 FAM208B CDC42EP2 CRELD2 MMD GPD2 CTSL1SLC1A4 SAA1 GTPBP4 DRAM1 ABHD14B ARAP2 TUBA4A TRIM2 PDK1 ANKRD22 SMCHD1NFKBIZ C9orf150 CDS1 FOXM1 ADAM8 MLLT3 ALS2CL SMARCC1 TRIB2 SSBP2 GALNT6TFDP1 GSDMB C3orf23 KRT15 XPO6 SGK1 DDT XDH TOP2B ARHGEF16 SARM1 DGAT2HEATR1 LRCH4 OSBPL6 FBP1 ODC1 HERC5 LEPREL2 CLDN10 ZWINT PLEKHM1EEF1A1P5 RHOD ATXN10 ASAH1 HCN2 PRSS3 NUP155 TMEM8A VLDLR CTSL2 CDCA7LPRICKLE4 BAMBI SNAI2 SNRPB NEU1 USE1 GALNT12 GNPNAT1 TNFSF15 CGREF1P2RY2 WDR3 PLAUR ATHL1 RAG1 HJURP AC103810.2 GPR37 PADI3 KNTC1 RHBDF1DERL3 TESC REV3L HSD17B11 PLK3 SELL CDH2 ROBO4 SLC29A4 GPR116 FARSB EREGTMEM234 GCHFR CCDC88A VWA1 RP11- RP11- 66N24.3.1 314P12.3.1 NETO2 TJP3CORO6 CHST4 NUP153 PDCD4 C9orf37 SHANK2 USP1 PTPRE EEF1A1P6 DNAH10 MCM6C15orf48 DNAJC3-AS1 KRT23 POLD2 TPBG C3orf78 COL1A1 SF3A3 TNFAIP3 MCEENMNAT2 TYMS PROS1 RP11- AKAP6 480A16.1.1 KIAA1324 GPR108 ZNF70 AADACTMEM48 AGFG2 NAT6 VEPH1 TCOF1 RHBDD2 SEMA3F C7orf58 PHIP DGAT1 VKORC1DNAH12 BRIX1 HBP1 RP5- SLC22A20 1103G7.4.1 UTP20 RNF103 P2RX6 MCC CTPSTSC22D3 C10orf102 RP11- 7K24.3.1 RANBP1 CAPS THTPA ANKRD56 SACS SLC2A6FTX CTD- 2021J15.2.1 DTL ABCA7 AC002472.8.1 ARL14 CDC20 TMC4 RP11-C15orf62 243J18.3.1 AP1B1 FAM193B CTD- METTL7B 307407.5.1 CDC6 VPS28RP11- SLC4A3 574K11.20.1 ABCD3 ARSD CES4A KLHL13 EBNA1BP2 NCF2 METTL12RP11- 357H14.19.1 PHTF2 MMP28 RP11- FGFR3 390P2.4.1 TRIP13 PARP10 RP11-PKDCC 216F19.1.1 RUVBL1 LZTS2 RP11- RP11- 85K15.2.1 157P1.4.1 RCC1ANKRD13D SEPT7L SMO MYBL2 DNASE2 CD22 CHML LYNX1 SYTL5 CCDC165 APOBEC3GTNNT2 SEH1L CDH3 ADAMTS14 WDR12 AQP3 ANXA10 TMEM56 NYNRIN TTC3P1 ERC1ABCG1 NMU RFC3 SH3YL1 RP11- 597D13.9.1 DEPDC1 FBXO2 UNC5C RP1- PPP1R16AAADAT 239B22.1.1 BMP2K SESN3 GRHL1 NT5DC2 TMPRSS3 TMEM169 MPHOSPH9HEATR7A CYP24A1 HDAC9 KYNU RP11- 448G15.3.1 STMN3 METTL7A ASB9 MRTO4FA2H NAALADL2 CDCA8 ZC3H12A FAM46B S100A2 CFI CFTR PI4K2B SLC15A3C16orf74 MCM8 SIGIRR RP11- 416I2.1.1 GTF2H3 FER1L4 PLAC1 JUN SLC39A11ZNF658 TTF2 FOXQ1 SP6 FAM171A1 RASSF5 TTN FKBP5 DHRS3 C8orf46 ADSLARHGEF3 GPR65 DIAPH3 S100P LEF1 KIAA0586 ZG16B RP11- 6F2.4.1 POLQ PTK6DENND2A EXO1 CDA EEF1E1 IL32 ODZ3 ZNF862 SSX2IP CD14 CEP97 TMED1 ABCC4G0S2 RAD51AP1 ENDOV MRPL47 SMAD6 C9orf140 MIA GEMIN5 CACNB1 FANCD2 ARL4DCCNE2 JAK2 UMPS KCNMB4 BARD1 IRAK2 USP13 RELB CENPV CEBPB SMTN MAFFPRPS1 C9orf16 CCNF RALGPS1 RFC5 LTB4R ATP5G1 CSF2RA POLR3G C10orf32 CBX2SAT2 NOP16 ALPPL2 MCM10 CCDC69 SLC16A7 NR1H3 POP1 GRAMD1C CLSPN FAM113BPFAS WASH7P KLF12 TGFBR3 GEMIN4 YPEL3 C3orf26 LRRN1 GMNN MAST3 CENPNABC7- 42389800N19.1.1 KIF15 ZNF467 GDAP1 XAF1 THOC7 C16orf7 OPN3 RP3-395M20.8.1 C22orf29 DNAH2 BAG2 ADCY4 ERCC2 CDC42EP5 JAM3 ITGAX DERAANKRD42 GPRIN1 NLRP1 E2F2 AC021593.1 CDC45 N4BP2L1 ARRB2 CLDN15 NCS1PIWIL4 SFMBT1 S1PR1 RAD54L HLA-H C1orf112 NEURL3 PRKAR1B P2RX4 FANCMAC007283.5.1 CENPL CA11 SELRC1 DPEP1 DOCK10 TBC1D3F ORC1 IER5L ALDH1B1PER1 BCL2 ZSWIM4 PSMC3IP ITGA10 SNRNP25 IL15 PRIM1 SERPINA3 HSPA4L RTP4SUV39H2 GLI4 MAGOHB C17orf103 HMGB1P5 SIX5 RP11- SNPH 14N7.2.1 WDR77PIP5K1B CENPH TFEB NME1 IDUA LRRC8C TMEM102 AC027612.6.1 SPRY3 METTL8FMO5 POLR1E MTRNR2L9 HOMER1 VSIG10L RGS17 GIMAP2 DCLRE1B PLCH2 POLE2LRRC6 CENPW LRFN3 SKA1 RILPL2 ADAT2 TNFSF12 CCDC41 CROCC WDR4 HLA-KCCDC18 PRICKLE3 PDSS1 CXCL3 C12orf24 NOS3 PARVB FBXO6 MMP24 CCDC146C14orf126 CSF2 SLC25A15 CEP85L NEGR1 GEMIN8 CAP2 ZNF628 OIP5 CXCL2 KIF5CZDHHC1 ARHGAP11B NR3C2 FAM86A RP11- 285F7.2.1 TBC1D7 PLEKHF1 CEP128RP11- 403I13.8.1 DLEU2 B3GALT4 BOLA3 NEIL1 COQ3 KNDC1 C17orf89 PON3ARHGEF26 CFB GDF11 ENDOU ACTR3B FOXO4 ASRGL1 DDO XYLB BBC3 RP1- KB-140K8.5.1 1460A1.5.1 SFXN2 SLC16A13 TIMM8A GAS7 MNS1 RNF152 GTF2H2 ABCA6AC009948.5.1 SLC16A6 ZNF239 RENBP RP11- KIAA1683 253E3.3.1 ASTN2 BBS12CHAC2 CACNA2D4 CSMD3 UPK3B RASSF2 AP001372.2.1 CCDC138 ZNF517 C1orf74ACYP2 CCDC134 ARHGAP25 CABYR PRX RP6- CES3 65G23.3.1 PPIAP29 ACCN3CYB5RL C19orf51 RP11- IL4I1 521B24.3.1 RP1- VMAC 152L7.5.1 C20orf94TNFRSF9 AL357673.1 RP11- 757G1.6.1 ISPD NFAM1 ELOVL2 RP11- 353B9.1.1RBM24 PDZD7 RP11- CARD14 204J18.3.1 DPF1 NYAP1 RP11- CD34 381E24.1.1RP11- RP11- 1334A24.4.1 325F22.3.1 MIR621 EXOC3L4 AC092329.1 RP5-1182A14.3.1 RP3- JAK3 324O17.4.1 CTD- WDFY3-AS2 2574D22.2.1 PDE1A PDGFRARP11- MLXIPL 33N16.1.1 RP11- KLHDC1 618G20.2.1 MIR29C RP11- 712B9.2.1CTD- 2292P10.4.1 PCSK4 CLDN9 PODN COL11A2 RP11- 65J3.1.1 PPP1R32 EFHC2TMEM105 AC005152.2.1 CASP5 TBX6 DNAH10OS SCARF1 RP11- 420G6.4.1 ROM1PYGM SLCO4C1 CCDC114 FAM71E1 RP11- 263K19.6.1 CTD- 2341M24.1.1 PCDHAC1CLIP3 C7orf63 C17orf108 ECEL1P2 LRRC24 ZNF385C TPPP3 RP11- 108M9.4.1ANKRD24 SRCRB4D MIR29B2 DNAH7 RP11- 454H13.6.1 RP11- 369J21.5.1 TMPRSS5PTPRCAP BCO2 C2orf81 FAM66C LINC00176 GPR132 SLC5A12 PDE4C ICAM5C20orf195 FBXO24 NGFR TNFSF13B MST1P9 CH25H CTC- 523E23.1.1 NFE2 CTC-378H22.2.1 FAM132A GBP5 HOXD4 UCN NKPD1 ELF5 LINC00324 RP11- 115C10.1.1ANGPTL1 RP11- 536G4.2.1 FAM66D RP4- 541C22.5.1 HSPA6 CTD- 2313N18.5.1CTD- 2547L24.3.1 CYP7A1 IGFALS RRM2P3 U7

Although the invention has been described with reference to the aboveexample, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1-12. (canceled)
 13. A method for identifying a subject's responsivenessto a treatment regime comprising analysis of changes in global chromatinmodifications in a DNA sample from the subject prior to and followingthe treatment regime to provide a prognosis or analysis ofresponsiveness to the treatment regime.
 14. A The method of claim 13,wherein identifying changes in global chromatin modification comprisesidentifying changes in large-organized heterochromatin lysine (K)-9modified domains (LOCKs), changes in large LOCKs, and/or changes ineuchromatin domains (ECDs).
 15. The method of claim 14, whereinanalyzing is by combinatorial methods.
 16. (canceled)
 17. The method ofclaim 15, wherein combinatorial methods comprise Western blotting,immunohistochemistry, chromatin immunoprecipitation (ChIP), whole genomebisulfite sequencing, or a combination thereof.
 18. The method of claim13, wherein the sample is DNA from a cancer cell from a solid tumor. 19.The method of claim 1, wherein the solid tumor is pancreatic ductaladenocarcinoma (PDAC).
 20. The method of claim 14, wherein identifyingchanges in LOCKs comprises identifying changes in H3K9Me2 and/orH4K9Me3.
 21. The method of claim 20, wherein detecting a loss of H3K9Me2in LOCKs in a cell from a primary tumor or from a local metastasis isindicative of a likelihood of evolution to distant metastasis.
 22. Themethod of claim 14, wherein identifying changes in ECDs comprisesidentifying changes in H3K26Me3 and/or H3K27Ac.
 23. The method of claim22, wherein detecting local reprogramming of H3K26Me3 and/or H3K27Acover differentially expressed genes withing ECDs in a cell from aprimary tumor or from a local metastasis is indicative of a likelihoodof evolution to distant metastasis.
 24. The method of claim 14, whereina LOCK has a length ranging from about 100 kb to 30 Mb.
 25. The methodof claim 14, wherein a LOCK has an average length of about 232 Kb-311Kb.
 26. The method of claim 14, wherein a large LOCK has a lengthranging from about 500 Kb to 30 Mb.
 27. The method of claim 14, whereina large LOCK has an average length of about 73 0 Kb-1,340 Kb.
 28. Themethod of claim 14, wherein an ECD has a length ranging from about 50 kbto 12 Mb.
 29. The method of claim 14, wherein a LOCK has an averagelength of about 207 Kb-277 Kb.